THE COMPOSITION AND ANTIMICROBIAL ACTIVITY OF LEAF

THE COMPOSITION AND ANTIMICROBIAL

ACTIVITY OF LEAF ESSENTIAL OILS OF SELECTED

AGATHOSMA SPECIES (RUTACEAE)

Carla Fourie

(Student number: 0111602D)

A research report submitted to the faculty o f Health Sciences, University o f the

Witwatersrand, Johannesburg, in partial fulfillm ent o f the requirements fo r the

degree o f Master o f Science in Medicine (Pharmaceutical Affairs).

Johannesburg, 2003

Declaration

I, Carla Fourie declare that this research report is my own work. It is being submitted for the degree o f MSc (Med) Pharmaceutical Affairs in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University.

it . .ih

day of N w jp fth r.... , 2003

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Acknowledgements

I would like to thank the following:

My supervisors Dr A. Viljoen and Ms S. van Vuuren.

My husband, Mome.

Prof Ba§er, Drs Bettil Demirci and Temel Ozek from the Medicinal and Aromatic Plant and Drug Research Centre, University of Anadolu, Turkey for the hospitality and efficient help with the GC-MS analysis during my visit to the Research Centre.

Ms Janine Victor of the National Botanical Institute and Mr Trinder-Smith (Bolus Herbarium) for assisting in the collecting and identification o f the various plant specimens.

The National Research Foundation, University o f the Witwatersrand Research Committee and Faculty of Health Sciences Research Endowment fund for financial support.

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Table of contents

List of figures 6

List of tables 8

Abstract 9

1. Introduction:

1.1 History 10

1.2 Essential oils 11

1.3 Microbiological activity o f essential oils 12

1.4 The Rutaceae 14

1.5 The genus Agathosma 15

1.6 Previous research on Agathosma 16

2. Material and methods

2.1 Collection o f plant material 20

2.1.1 Extraction o f essential oils 20

2.2 Antimicrobial testing 21

2.2.1 Test organisms 21

2.2.2 Disc diffusion assay 21

2.2.3 MIC/microplate bioassay method 23

2.3 Analytical chemistry 25

2.3.1 Thin-layer chromatography (TLC) 25

2.3.2 Gas chromatograph-Mass Spectrometry (GC-MS) 25

2.3.3 TLC bioautographic assay 25

3. Monographs of Agathosma species studied

Agathosma arida 26

Agathosma capensis 29

Agathosma lanata 34

Agathosma mundtii 37

Agathosma ovalifolia 40

4

Agathosma ovata 43

Agathosma recurvifolia 47

Agathosma serpyllaceae 50

Agathosma zwartbergensis 53

4. Results and discussion

4.1 Antimicrobial activity 55

4.2 Analytical chemistry 62

4.3 Antimicrobial activity of main compounds 65

5. Conclusion 68

6. References 69

5

Figure 1: Agathosma ovata (George Botanical Garden).

Figure 2: Charging the stills with plant material.

Figure 3: Preparation of plates for disc diffusion method by pouring base layer and

proceeding with inoculated top layer.

Figure 4: Aseptic transfer of discs with Agathosma essential oil onto seeded agar plate.

Figure 5: Preparing the MIC plate by performing doubling dilutions of Agathosma oil.

Figure 6: Serial dilutions of the essential oils of A. capensis (Gamka), A. ovata (Gamka),

A. ovata (Anysberg) and A. recurvifolia.

Figure 7: Geographical distribution of A. arida.

Figure 8: Gas chromatography profile of A. arida.

Figure 9: Chemical structures of the major compounds identified in A. arida essential oil.

Figure 10: Geographical distribution o f A. capensis.

Figure 11: Gas chromatography profile o f A. capensis (Gamka).

Figure 12: Gas chromatography profile of A. capensis (Rooiberg).

Figure 13: Gas chromatography profile o f A. capensis (Mossel Bay).

Figure 14: Chemical structures of the major compounds identified in A. capensis essential oil.

Figure 15: Geographical distribution of A. lanata.

Figure 16: Gas chromatography profile o f A. lanata.

Figure 17: Chemical structures of the major compounds identified in A. lanata essential oil.

Figure 18: Geographical distribution o f A. mundtii.

Figure 19: Gas chromatography profile of A. mundtii.

Figure 20: Chemical structures of the major compounds identified in A. mundtii essential oil.

Figure 21: Geographical distribution o f A. ovalifolia.

Figure 22: Gas chromatography profile of A. ovalifolia.

Figure 23: Chemical structures of the major compounds identified in A. ovalifolia essential

oil.

List of Figures

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Figure 24: Geographical distribution of A. ovata.

Figure 25: Gas chromatographic profile of A. ovata (Gamka).

Figure 26: Gas chromatographic profile of A. ovata (Anysberg).

Figure 27: Chemical structures of the major compounds identified in A. ovata essential oil.

Figure 28: Geographical distribution of A. recurvifolia.

Figure 29: Gas chromatographic profile of A. recurvifolia.

Figure 30: Chemical structures of the major compounds identified in A. recurvifolia essential

oil.

Figure 31: Geographical distribution of A. serpyllacea.

Figure 32: Gas chromatographic profile of A. serpyllacea.

Figure 33: Chemical structures of the major compounds identified in A. serpyllacea essential

oil.

Figure 34: Geographical distribution of A. zwartbergensis.

Figure 35: Gas chromatographic profile of A. zwartbergensis.

Figure 36: Chemical structures of the major compounds identified in A. zwartbergensis

essential oil.

Figure 37: Disc diffusion plate of E. coli on the essential oils of Agathosma species studied.

Figure 38: Disc diffusion plate of B. cereus on essential oils of (34-^4. capensis (Gamka); 36

-A . ovata (Gamka); 38 - A. zwartbergensis', 40 -A . ovalifolia).

Figure 39: MIC results after 24 hours.

Figure 40: TLC bioautographic assay.

Figure 41: TLC plate of Agathosma essential oil. The plate has been sprayed with vanillin-

sulphuric acid.

Figure 42: TLC plate of Agathosma essential oil. The plate has been sprayed with

anisaldehyde-sulphuric acid.

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List of Tables

Table 1: List of study samples and percentage oil yield after distillation.

Table 2: GC-MS results ofvl arida.

Table 3: GC-MS results of A. capensis (Gamka) - A, A. capensis (Rooiberg) — B and A.

capensis (Mossel Bay) - C.

Table 4: GC-MS results o f A. lanata.

Table 5: GC-MS results of A. mundtii.

Table 6: GC-MS results of A. ovalifolia.

Table 7: GC-MS results of A. ovata (Gamka) - A and A. ovata (Anysberg) - B.

Table 8: GC-MS results of A. recurvifolia.

Table 9: GC-MS results of A. serpyllacea.

Table 10: GC-MS results of A. zwartbergensis.

Table 11: Antibacterial screening results as expressed in the disc diffusion assay (mm from

disc edge).

Table 12: Antifungal disc diffusion screening results (expressed as mm from disc edge).

Table 13: MIC results after 30 minutes, 2 hours and 24 hours.

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AbstractThis project was conducted to investigate the antimicrobial properties and to record

the essential oil profiles o f a selection o f species belonging to the genus Agathosma.

Plants have been used for many years by the local South Africans to treat various

infections and illnesses. This knowledge has largely been untapped. Buchu is one of

the plant species that are used extensively by the San and Khoi people. It is

remarkable that o f the ca. 150 Agathosma species indigenous to South Africa only

two species (Agathosma crenulata and Agathosma betulina) have been investigated

for biological activity. The genus Agathosma is traditionally used for the following

conditions; stomach ailments, fever, coughs, colds, flu, urinary tract and kidney

infections, haematuria, prostatitis, rheumatism, gout, bruises and for antiseptic

purposes.

The antimicrobial activity and leaf essential oil chemistry were investigated for A.

arida, A. capensis, A. lanata, A. mundtii, A. ovalifolia, A. ovata, A. recurvifolia, A.

serpyllaceae and A. zwartbergensis. The phytochemistry o f the essential oils was

analyzed by using gas chromatography-mass spectrometry. The antimicrobial

properties were analyzed by using disc diffusion assays, MIC/microplate assays and

TLC bioautographic assays.

All the species showed some degree o f antimicrobial activity. The minimum

inhibitory concentrations o f A. capensis, A. ovata and A. recurvifolia were

determined. A TLC bioautographic assay for A. zwartbergensis indicated that

citronellal could be responsible for the antimicrobial activity.

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1. Introduction:

1.1 HistoryHistorically, plants have been a source o f inspiration to scientists searching for novel

drug compounds and have made large contributions to human health. Natural

compounds from plants may become a natural blue print for the development o f new

drugs or they may be used in the crude form for the treatment o f disease based on

their application by traditional societies from different parts o f the world. Today it is

estimated that plant products have contributed to 50% o f Western drugs. The primary

benefits o f using plant-derived medicines are that they are relatively safe, offer

profound therapeutic benefits and are more affordable (Iwu et al., 1999).

Microbiologists have two reasons to be interested in the topic o f antimicrobial plant

extracts. Firstly, it is likely that these phytochemicals may be used, by physicians, as

antimicrobial drugs and secondly, the public is becoming increasingly aware o f the

problems with over prescribing and misuse o f antibiotics. Another driving factor for

the renewed interest in plant antimicrobials has been the rapid rate o f plant species

extinction. The use of essential oils for healing purposes has been known in

traditional medicine. It is still o f interest today because o f the trend back to natural

drugs and therapies in medicine (Cowan, 1999).

Aromatherapy has been practiced for centuries. The Greeks, Romans and Egyptians

all used aromatherapy oils. Hippocrates, the father o f modem medicine, used

aromatherapy baths and scented massage. The modem era o f aromatherapy dawned

in 1930 when the French chemist Rene Maurice Gattefosse used the term

aromatherapy for the therapeutic use o f essential oils. He began his research into the

healing powers o f essential oils after an accident in his laboratory when he burned his

hand. He immersed his injured hand in a vat o f lavender oil (containing linalool as

major component) and was quite impressed with the healing results. He started

investigating further healing properties o f essential oils

(www.naturalaromatherapy.com).

Commonly known herbs like cinnamon, ylang-ylang, basil, lemongrass, marjoram and

rosemary are all essential oil containing plants. The antimicrobial and antioxidant

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http://www.naturalaromatherapy.com

properties o f these herbs together with lemon fruit were studied by Baratta et al.

(1998). Generally all the oils, exhibited antibacterial activity.

One o f the more recent success stories o f essential oil research is that o f Tea Tree oil

obtained from Melaleuca alternifolia. For thousands o f years the native Aborigines o f

Australia have used the leaves o f the Tea Tree to cure various ailments. Early in this

century, doctors and scientists began to realize that the natural oil contained in the

leaves has healing properties. Today Tea Tree oil is recognized as an extremely

effective curative for a wide range o f common medical conditions (Combest, 2000).

1.2 Essential oils

Plants have a limitless ability to synthesize aromatic substances. Essential oils are

volatile in steam and are generally mixtures o f hydrocarbons and oxygenated

compounds. The oxygenated compounds determine the odor and taste o f volatile oils

(Evans, 1996). These oils are secondary metabolites (compounds) based on an

isoprene structure. These oils are called terpenes and occur as monoterpenes (Cio),

sequiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40), as well

as hemiterpenes (C5). When these compounds contain additional elements, usually

oxygen, they are termed terpenoids (Cowan, 1999). Terpenoids comprise the largest

organic chemical group, not only in essential oils, but also in natural products.

Essential oils also contain other classes o f molecules including phenolics, aromatics,

cyclic and acyclic compounds, acetonides, and sulphur- and nitrogen-containing

compounds, depending on the plant and the extraction method (Nakatsu et a l, 2000).

Terpenes are active against bacteria and fungi. The mechanism o f action is not fully

understood but it might involve membrane disruption by the lipophilic compounds

(Cowan, 1999). In general, gram-negative bacteria have been found to be more

resistant to essential oils than gram-positive bacteria. Mangena and Muyima (1999)

state a reason in an article on comparative evaluation o f the antimicrobial activities o f

essential oils o f Artemisia afra, Pteronia incana and Rosmarinus officinalis on

selected bacteria and yeast strains, and can be attributed to the lipopolysaccharide cell

wall o f the gram-negative bacteria.

Essential oils (they are called essential because o f the previous belief that each oil

represented the essence o f the original plant) o f aromatic plant species are used in

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industry for the production o f soaps and perfumes. In view o f the increasing use o f

essential oils in the food, cosmetic and pharmaceutical industries, it is important to

examine the oils from indigenous plants for antimicrobial activities. Essential oils

have been used as medicine since ancient times and form part o f traditional folk

medicine. Therefore they are considered to be the most widely used natural products

(Nakatsu et al., 2000).

1.3 Microbiological activity of essential oils

Testing and evaluating antimicrobial activity o f essential oils is difficult because o f

their volatility, their water insolubility and complexity. Janssen et al. (1986) mentions

that the following factors may change the composition o f essential oils: the botanical

source, the provenience of the plant material, the condition o f the plant material (fresh

or dried) and the isolation technique (steam distillation or hydrodistillation). These

factors must be taken into consideration when evaluating the antimicrobial activity of

essential oils. The following factors also influence the results o f antimicrobial

susceptibility tests o f essential oils: methodology, the microorganisms and essential

oils used.

Buchbauer (1993), states that essential oils are heterogeneous mixtures o f single

substances. Hence one has to be aware that biological actions are due to this complex

mixture o f various aromatic chemicals. Each constituent contributes to the biological

effect in a complicated synergistic or antagonistic way.

Articles published by researchers in the last ten years, in the field o f essential oil

biological activity, focussed on the activity to inhibit microorganisms. Usually the

essential oil inhibition on bacteria and fungi is determined followed by an

investigation o f the activity o f the essential oil components. The most common

method used for antimicrobial assays o f essential oils is the disc diffusion method.

The most frequently effectively used method to separate and identify essential oil

components are by using gas chromatography coupled to mass spectrometry (GC-

MS). This method makes use o f database libraries o f both retention indices and mass

spectral fragmentation patterns (Nakatsu et ah, 2000).

The biological activity of spices and herbs has re-emerged as an area o f interest.

Thymus species is a common spice that has been extensively studied. The essential oil

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that is isolated from this spice is active against gram-positive and gram-negative

bacteria. The major constituent is thymol and has been implicated to be the molecule

responsible for the activity. Salvia species or more commonly known as sage shows

activity towards fungi and the major component that is responsible for the activity is

cineole. Rosemary and Origanum species show antimicrobial activity. Anethole,

limonene and fenchone found in fennel show antifungal activity. The major

constituent o f mint is menthol that plays a significant role in its activity towards

bacteria and fungi. Eugenol commonly found in clove inhibits the growth o f yeast

(Nakatsu et al., 2000).

The chemical composition and antimicrobial activity of the essential oil o f

Calamintha nepeta was investigated by Flamini et al. (1999). They found that the

essential oil showed strong activity against Salmonella species and noteworthy

effectiveness against mycetes parasites like Aspergillus niger (which can be

pathogenic for man both by its spores an by the production of mycotoxins). In their

investigations they also verified those constituents, which may be responsible for the

strong activity against Salmonella species. The main constituents limonene,

menthone, pulegone, and menthol were determined by using GC and of these the only

one showing antibacterial activity was pulegone.

The essential oil o f Crysanthemum viscidehirtum consists mainly o f limonene, (3-

famesene and many oxygenated sesquiterpenes. According to Khallouki et al. (2000)

this essential oil exhibits activity against particularly Salmonella typhi and Proteus

mirabilis.

Cobos et al. (2001) investigated the chemical composition and antimicrobial activity

of the essential oil of Baccharis notosergila. The chemical analysis was done by

using GC-MS and the main components were determined as a-pinene, limonene, (3-

caryophyllene and spathulenol. The antimicrobial activity was determined by using

two techniques: the well diffusion and the paper disc diffusion method. The more

susceptible strains were the gram-positive bacteria. Proteus mirabilis was the only

gram-negative bacteria that were inhibited by the essential oil and Candida albicans

demonstrated good sensitivity. They came to the conclusion that essential oils

containing monoterpenes like limonene are more active against gram-positive

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organisms and fungi than gram-negative organisms. They also found that spathulenol

was found to be active against Candida albicans and Proteus mirabilis.

Mangena and Muyima (1999), compared the antimicrobial activity o f Artemisia afra,

Pteronia incana and Rosmarinus officinalis, A. afra had a broad-spectrum

antibacterial activity and the main chemical constituents were Q*-thujone and (3-

thujone. P. incana displayed a fairly broad-spectrum antibacterial activity and the

main constituents were /3-pinene and opinene. R. officinalis had the same activity as

A. afra but the main constituents differ, being camphor, 1,8-cineole and a-pinene for

R. officinalis. A. afra seemed to produce larger zones o f inhibition than P. incana and

R. officinalis when the oils were tested on yeasts.

Composition and activity o f Salvia ringens essential oil was studied by Tzakou et al.

(2001) and the major constituents were 1,8-cineole, bomyl acetate, /3-pinene and a-

pinene. Their antibacterial results showed that S. ringens appeared to be inactive

against gram-positive bacteria (S. aureus and S. epidermidis) while it showed strong

activity towards gram-negative bacteria. They also screened the pure monoterpenoids

a-pinene and 1,8-cineole to compare the results with the investigated oil. They came

to the conclusion that the activity can be attributed to these two constituents especially

1,8-cineole.

1.4 The Rutaceae

The genus Agathosma belongs to the citrus family (Rutaceace). Trees, shrubs or

shrublets belonging to the family Rutaceace are usually aromatic plants. The family

is distributed in both temperate and tropical countries, but particularly abundant in

South Africa and Australia. Glands containing essential oils are present in the leaves

and other parts. The flowers are usually in cymes with 4-5 sepals, 4-5 petals, 8-10

stamens and a superior ovary. The fruits are o f various types. Constituents o f the

Rutaceace include a wide variety o f alkaloids, volatile oils, rhamno-glucosides,

coumarins and terpenoids (Evans, 1996).

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1.5 The genus Agathosma

The Cape region o f South Africa has veld-types with arguably the richest composition

o f indigenous aromatic plant species in the whole o f South Africa. Among these

aromatic plants is the genus Agathosma that is restricted to this region. These shrubs

are typical o f the fynbos vegetation and are found in mountainous areas in the Cape

(van Wyk and Gericke, 2000).

Agathosma species or commonly known as buchu, are perennial shrubs with woody

branches and small, flat, gland dotted leaves. The flowers are star-shaped and open.

The flowers contain five petals, five stamens, a five-lobbed ovary and the leaves have

a characteristic smell when crushed (van Wyk et al., 1997). The name buchu is

applied by the Hottentots to any aromatic herb or shrub. The Buchuberg in

Namaqualand does not derive its name from the genus Agathosma but from other

aromatic plants. In Griqualand West Othonna species are known as “buchu” and is

used as a cosmetic. Empleurum species commonly referred to as false buchu and berg

buchu has also been referred to in trade as “buchu”. The genus Diosma is often

referred to as “wild buchu” and is used only when true buchu is not available (Watt

and Breyer-Brandwijk, 1962).

Finding healing powers in plants is an ancient idea and so Buchu is an important part

of the Khoi culture in the Cape and is still used as a general tonic and medicine

throughout South Africa. It is also well documented for its cosmetic purposes.

A summary o f the traditional uses o f the genus Agathosma is listed below (Watt and

Breyer-Brandwijk, 1962);

• To cause febrifuge (profuse perspiration).

• As an antispasmodic.

• As an antipyretic.

• A cough remedy, as well as for colds and flu.

• Kidney and urinary tract infections, as well as for hematuria and protatitis.

• To relief rheumatism, gout and bruises.

• A sa diuretic and for genito-urinary system infections.

• Has been used as a liniment or embrocation.

• For relief o f calculus,

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• Is used for treatment o f cholera and other stomach ailments.

• Has been used for antiseptic purposes.

Agathosma species have been used for cosmetic purposes and as an antibiotic. The

leaves were used in a variety of preparations. The leaves were chewed or prepared in

a tincture containing brandy to relieve stomach complaints. A mixture o f buchu and

vinegar is still being used today to clean wounds (van Wyk et at, 1997). Boiling

water is poured over lg buchu leaves, covered and allowed to stand for 10 minutes

before being strained. A cup of the infusion is drunk several times a day as a diuretic

(Bisset, 1994).

Of the 150 species, Agathosma betulina (round-leaf buchu) and A. crenulata (oval-

leaf buchu) are mainly used for medicinal purposes. These two species are important

sources o f valuable essential oils (van Wyk and Gericke, 2000). The essential oil of

A. betulina is a dark yellow-brown oil with a minty-camphoraceous odour.

Agathosma betulina and A. crenulata are cultivated and are in the process o f being

developed as crop plants. These two species are the only two that have been used

commercially. This is due to the limited availability o f the wild plant material (van

Wyk and Gericke, 2000). Buchu forms part of about 10 prepared herbal teas,

including BuccoteanR Tee, BuccosperinR Tee, Uron-Tee tea bags and is a constituent

of the UK product Potter’s Kas-bah Herb. Other preparations available in the UK

containing buchu are: Potter’s Diuretictabs, Antitis Tablets, Backache Tablets,

Stomach Mixture, Gerard House Herbal Powder and Buchu Compound Tablets

(Bisset, 1994).

Agathosma serratifolia (narrow-leaf buchu) a willow-like small tree, A. pulchella and

A. ovata (false buchu, Figure 1) a small rounded shrub with pink flowers, have also

been used for medicinal purposes among the Cape people (van Wyk and Gericke,

2000).

1.6 Previous research on Agathosma

Buchu oil is a rarely studied oil and Lawrence (1976) summarized the different

studies, in Progress In Essential Oils, reprinted from Perfumer & Flavorist, until 1976.

In 1961 Fluck and coworkers succeeded in identifying pulegone and diophenol as

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constituents o f buchu oil. The first comprehensive analysis o f buchu oil was published

in 1968 by Klein and Rojahn in which they isolated and characterized seventeen

compounds. Lamparsky and Schudel isolated 8-mercapto-p-menthan-3-one from

Buchu oil in 1971 and found that this sulphur containing terpene was very important

for the flavour and aroma of the oil.

Figure 1: Agathosma ovata (George Botanical Garden).

The most detailed and thorough study was by Kaiser et al. (1975) on the analysis of

Buchu leaf oil. They identified more than 120 constituents including the already

known pulegone, diosphenol and 8-mercapto-/>-menthan-3-one in Buchu leaf oil of

commercial origin. Their study was conducted to determine its aromatic important

components. They determined that characteristic o f the oil is the occurrence of

bifimctional monoterpene ketones which can be classified as sulphurated and

oxygenated derivatives o f /?-menthan-3-one. The mixture o f 2-acetoxypulegones

gives buchu oil a minty, hay-like odour. They also acknowledge that sensoric

properties differ when comparing oils from different producers and materials of

different botanical origin. In their study they compared an oil distilled in South Africa

originating from Barosma betulina, an oil distilled in South Africa originating form

Barosma crenulata and a so-called “English distilled” oil. They concluded that the

more important aromatic 8-mercapto-/>-menthan-3-one is present in larger quantities

in Barosma betulina than in Barosma crenulata (Barosma is an old synonym of

Agathosma).

Since then only a few studies have appeared. Two articles were published in the

Journal o f Essential Oil Research (1996). The one article, by Posthumus et al. (1996)

17

was on the chemical composition o f the essential oils o f A. betulina, A. crenulata and

a Buchu hybrid. Chemical investigation was done by means o f chromatographic and

spectroscopic methods and their ultimate aim was to recognize plants with a specific

chemical composition. The results indicated that buchu contains a few common

monoterpenes and a number o f rare bi- and trifunctionalized monoterpenes. These

compounds included two diosphenols, hydroxylated diosphenols, several

hydroxymenthones and some acetates thereof. They made use o f the publication by

Kaiser et al. (1975) that contained the peak order and the chromatogram and they used

the highly characteristic mass spectra o f these constituents to identify them. Further

they identified in total 40 known compounds. According to their study A. betulina is

characterized by 31% of (iso)menthone, 41% (i/')-diosphenol and 3% cis- and trans-8-

mercapto-/>-menthane-3-ones, A. crenulata contains very high quantities o f pulegone

(54%) and the hybrid showed a high concentration o f (iso)menthone (55%) and

otherwise a intermediate composition. Collins et al. (1996) reported on the

chemotaxonomy of commercial Buchu species. The oils o f A. betulina and A.

crenulata and their hybrids were analyzed to determine i f they could be distinguished

by their monoterpene content. Agathosma betulina and A. crenulata are mainly

distinguished by their leaf form were A. betulina has more round leaves and is also

known as round leaf buchu and A. crenulata has more oval leaves. Hybridization has

occurred and has led to confusion in identification. Their study resulted in that all

three taxa contained the same constituents but in different percentages. Agathosma

betulina had high percentages o f limonene, menthone, isomenthone, (i^)-diosphenol,

diosphenol, cA-8-mercapto-p-menthan-3 -one, 4-hydroxy-diosphenol and 1-hydroxy-

diosphenol. Agathosma crenulata had a very high percentage o f pulegone and

isopulegone isomers and a higher percentage o f 8-acetylthio-p-menthan-3-one

isomers than A. betulina. The hybrids had the simultaneous presence o f high

concentration o f pulegone and diosphenol.

Round-leaf buchu contains a valuable essential oil rich in isomenthone and

diosphenol. Oval-leaf buchu yields a less desirable oil because o f the high levels o f

pulegone, a potentially toxic substance (van Wyk and Gericke, 2000).

Other components o f the essential oil o f Agathosma betulina are limonene, (-)-

isomenthone, (+)- menthone, (-)-pulegone, terpinen-4-ol and /;-menthan-3-on-8-thiol.

/>-Menthan-3-on-8-thiol is mainly responsible for the odour o f this species.

18

Diosphenol separates in its crystalline form at room temperature, which is called

buchu camphor. Unlike the round, serrulate leaves o f A. betulina, the leaves of other

Agathosma species contain little or no diosphenol. TLC and GLC can be used to

distinguish between the leaves of A. betulina and A. crenulata seeing that the

diosphenol zone or peak will be absent with the latter species (Bisset, 1994).

The main objective of this study is to investigate the possible antimicrobial properties

o f a selection of species belonging to the genus Agathosma (Rutaceae) and to record

the essential oil profiles. An attempt will be made to correlate the antimicrobial

activity to the essential oil composition.

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2. Material and Methods

2.1 Collection of plant material

Agathosma species are indigenous to the Cape Province o f South Africa. All

specimens were collected from the wild and voucher specimens and are housed in the

Bolus Herbarium, University o f Cape Town and at the National Herbarium in

Pretoria.

Table 1: List o f study samples and percentage oil yield after distillation.

Species Locality Voucher Yield (%)A. arida Rooiberg TTS-241 0.61

A. capensis Gamka TTS-243 1.76

A. capensis Rooiberg JEV-5 0.74

A. capensis Mossel Bay JEV-7 0.68

A. lanata Rooiberg TTS-242 0.37

A. mundtii Rooiberg TTS-238 0.27

A. ovalifolia Droekloof TTS-260 1.02

A. ovata Gamka TTS-246 0.22

A. ovata Anysberg TTS-263 0.21

A. recurvifolia Rooiberg TTS-240 0.14

A. serpyllaceae Heuningberg JEV-11 0.31

A. zwartbergensis Swartberg TTS-257 1.78

Mr. Trinder-Smith, from the University o f Cape Town, who is currently completing a

PhD on the taxonomy o f the genus Agathosma, confirmed the identity o f all

specimens.

2.1.1 Extraction of essential oils

Conventional hydrodistillation (Figure 2) was carried out using a Clevenger-type

apparatus to selectively extract the essential oils from the plant material. The plant

material is weighed before distillation and the amount of oil is weighed after

distillation to determine the yield o f oil. The essential oils are stored and refrigerated

in amber vials to minimize stability problems often encountered with essential oils.

20

2.2 Antimicrobial testing

2.2.1 Test organisms

Selection o f the test organisms took place by taking the traditional uses o f Agathosma

into consideration. Agathosma is used traditionally to relieve stomach complaints and

for minor digestive disturbances, therefore E. faecalis, E. coli, B. cereus and S.

typhimurium were selected. Buchu is used for washing and cleaning wounds,

therefore S. aureus and P. aeruginosa were selected. Buchu is also used to treat

kidney and urinary tract infections, hence C. albicans and E. coli were selected as

pathogens.

Bacterial test organisms were as follow: Staphylococcus aureus (ATCC 25923),

Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 9027),

Escherichia coli (ATCC 8739), Bacillus cereus (ATCC 11778) and Salmonella

typhimurium (clinical strain).

Fungal test organisms were as follow: Candida albicans (ATCC 10231),

Cryptococcus neoformans (ATCC 90112) and Aspergillus niger (clinical strain).

2.2.2 Disc diffusion assay

Four large Mueller-Hinton (Oxoid) agar plates were prepared containing 100 ml of

agar and 100 ml o f overlayed agar. Overnight broth cultures o f the following bacteria;

Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus

faecalis, with an inocculum size lxlO 6 was used to seed the agar. The bacterial spore

suspensions were incorporated into the top agar layer. Smaller agar plates (Figure 3)

were prepared for Salmonella typhimurium and Bacillus cereus. The smaller round

petri dishes contained 15 ml of agar and 15 ml o f agar seeded with inoculum.

Aseptic techniques were used to saturate discs (Figure 4) with essential oils and then

placed onto the seeded agar plates. A disc containing Neomycin 30 pg, (Oxoid) was

used as a positive bacterial control. The screening plates were refrigerated for 1 hour

to allow pre-diffusion and then incubated at 37 °C for 24 hours, after which zones of

inhibition were measured.

21

Figure 4: Aseptic transfer of discs with Agathosma essential oil onto seeded agar plate.

Figure 5: Preparing the MIC plate by performing doubling dilutions o f Agathosma oil.

Figure 2: Charging the stills with plant material. Figure 3: Preparation of plates for discdiffusion method by pouring base layer and proceeding with inoculated top layer.

A second series o f fungal test organisms namely Candida albicans, Cryptococcus

neoformans and Aspergillus niger were also studied. A disc containing Nystatin (100

lp., Oxoid) was used as a positive control. Tryptone Soya (Oxoid) agar was used for

the fungi and the same process as described above was followed to prepare the agar

and the saturated disc.

2.2.3 MIC/microplate bioassay method

After antimicrobial activity was recorded by the disc diffusion assay, the minimum

inhibitory concentrations (MIC) o f the most active oils were determined by using the

/i-iodonitrotetrazolium violet (INT) microplate method. A fixed bacterial culture

yielding an inoculum size o f lxlO6 was added to all wells. Further more, this method

involves dilution of the essential oils in microplate test conditions. An essential oil

concentration o f 32 mg/ml was prepared in the first row o f wells in the microplate

(Figure 5). Serial dilutions were performed (Figure 6). The addition o f the oils to the

microplate as well as the serial dilutions took place under laminar airflow to minimize

contamination.

Minimum inhibitory concentration (MIC) o f the following essential oils; A. ovata

(Gamka and Anysberg), A. capensis (Gamka) and A. recurvifolia, were determined for

Staphylococcus aureus, Enterococcus faecalis and Escherichia coli.

These oils and bacteria were chosen using the screening results as a guide to their

positive antimicrobial properties. Overnight broth cultures o f these organisms were

prepared as for the disc diffusion assay. A fixed bacterial culture yielding an inoculum

size o f lxlO6 was added to all wells. The microplate (Figure 5) was incubated for 24

hours at 37°C. Forty microliters o f INT solution, with a concentration o f 0.2 mg/ml,

was added to all wells after incubation. INT binds in a complex manner with the

DNA in bacterial cells. The INT solution is used as a bacterial growth indicator. The

pink colour change was observed after 30 minutes, 2 hours and 24 hours. The

minimum inhibitory concentration, o f each sample o f hydrodistilled oil, was

calculated. Minimum inhibitory concentrations were determined presumptively as the

first well, in ascending order, which did not produce a colour change.

23

1 2 3 4 5 6 7 8 9 1011 12AO o o o o o o o o o o oBO o o o o o o o o o o oCO o o o o o o o o o o oDO o o o o o o o o o o oeO o o o o o o o o o o oFO o o o o o o o o o o oGO o o o o o o o o o o oHO o o o o o o o o o o o

Column 1: A. capensis (Gamka) and Escherichia coli Column 2: A. capensis (Gamka) and Staphylococcus aureus Column 3: A. capensis (Gamka) and Enterococcus faecalis Column 4: A. ovata (Gamka) and Escherichia coli Column 5: A. ovata (Gamka) and Staphylococcus aureus Column 6: A. ovata (Gamka) and Enterococcus faecalis Column 7: A. ovata (Anysberg) and Escherichia coli Column 8: A. ovata (Anysberg) and Staphylococcus aureus Column 9: A. ovata (Anysberg) and Enterococcus faecalis Column 10: A. recurvifolia and Escherichia coli Column 11: A. recurvifolia and Staphylococcus aureus Column 12: A. recurvifolia and Enterococcus faecalis

Row A: Concentration o f essential oil = 32 mg/ml Row B: Concentration o f essential oil = 16 mg/ml Row C: Concentration o f essential oil = 8 mg/ml Row D: Concentration o f essential oil = 4 mg/ml Row E: Concentration o f essential oil = 2 mg/ml Row F: Concentration o f essential oil = 1 mg/ml Row G: Concentration o f essential oil = 0.5 mg/ml

Row H: Concentration o f essential oil = 0.25 mg/ml

Figure 6: Serial dilutions o f the essential oils o f A. capensis (Gamka), A. ovata (Gamka), A. ovata (Anysberg) and A. recurvifolia.

2.3 Analytical chemistry

2.3.1 Thin layer chromatography (TLC)

Silica thin layer plates were used together with toluene-ethyl acetate (93:7) as mobile

phase. Detection was made possible with the use o f spray reagents namely vanillin-

sulphuric acid and anisaldehyde-sulphuric acid.

After development, the TLC plates were air-dried and sprayed with one o f the

reagents. Colour development took place after a short heating period.

2.3.2 Gas chromatograph-Mass Spectrometry (GC-MS)

Essential oils were analyzed using the following operating conditions: Column: HP-

Innowax (60 m x 0.25 mm id., 0.25 jum film thickness), Temperatures: injection port

250 °C, column 60 °C for 10 min., 4 °C / min. to 220 °C, 220 °C for 10 min., 1 °C /

min. to 240 °C (total = 80 min.). Helium as carrier gas.

0.9 ill o f hexane with 0.1 /zl o f the essential oil was injected. Identification took place

with the use o f TBAM’s database libraries by matching both retention indices and

mass spectral fragmentation patterns. This part o f the project was completed by

myself at the Medicinal and Aromatic Plant and Drug Research Centre, Anadolu

University, Turkey.

2.3.3 TLC bioautographic assay

The hydrodistilled oil o f Agathosma zwartbergensis was chosen for the TLC

bioautographic assay. A Bacillus cereus spore suspension with an inoculum size of

lxlO6 was incorporated into Mueller Hinton agar. TLC plates o f the essential oil and

of a standard o f the main compound were developed and placed directly onto the

prepared agar plate. The TLC plates were developed by using toluene-ethyl acetate

(93:7) as the mobile phase and vanillin-sulphuric acid spray reagent to detect the

compounds o f the oil. The TLC overlay-agar plate was incubated for 24 hours.

25

3. Monographs of Agathosma species studied

Agathosma arida P.A. Bean

1. Botanical description

Single-stemmed, rounded shrublet to 40cm, sweetly herb-scented. Flowers in

terminal clusters, pink or violet. Fruits: 3-chambered. Ovary: usually 3-lobed.

2. Distribution

Gravelly loam, karoo-fynbos ecotone. This species is restricted to the Little Karoo,

specifically the northern slopes of Langeberg and Outeniqua Mountains (Goldblatt

and Manning, 2000).

Figure 7: Geographical distribution of A. arida.

3. Essential oil composition

Figure 8: Gas chromatography profile o f A. arida.

26

Table 2: GC-MS results o f A. arida.

R R I*R eten tio n tim e

(m in u tes)A rea

p ercen ta g eC o m p o u n d

1032 8.57 1.80 a-pinene1035 8.70 0.10 a-thujene (not integrated)1076 10.30 0.01 camphene1118 12.20 12 .28 3 -p in en e1132 12.86 3.98 sabinene1174 14.98 16.35 m yrcen e1188 15.65 0.10 a-terpinene1203 16.57 2 1 .7 9 lim on en e1218 17.00 0.83 P-pheilandrene1246 18.32 0.07 (Z)-p-ocimene1255 18.76 0.29 y-terpinene1266 19.07 0.25 (E)-P-ocimene1280 19.91 0.82 p-cymene1290 20.42 0.10 terpinolene1319 21.50 0.04 (E)-2,6 dimethyl 1,3,7 nonatriene1337 22.20 0.09 geijerene1429 25.97 0.07 perillen1430 26.17 0.03 a-thujone1451 26.88 0.21 3-thujone1468 27.52 0.38 trans- 1,2-limonene epoxide1495 28.27 0.06 bicycloelemene1490 28.58 0.02 isogeijerene C1506 28.91 0.15 decanal1541 29.91 0.01 benzaldehyde1553 30.40 11.02 lin a loo l1562 30.63 1.74 isopinocamphone1571 31.01 0.15 PwJs-p-menth-2-en-1 -ol1586 31.38 0.07 pinocarvone (not pure)1594 31.77 0.07 Pww-p-bergamotene (not pure)1611 32.33 2.49 terpinen-4-ol1626 32.75 0.47 2-methyl-6-methylene-3,7-octadien-2-ol1639 33.02 0.15 tra«s-p-menth-2,8-dien-1 -ol1645 33.27 0.08 c/j-isodihvdrocarvone1657 33.65 0.35 umbeilulone1678 34.32 0.04 cts-p-menth-2,8-dien-1 -ol

1687+ 34.56 0.76 a-humulene+methylchavicol1700 34.86 0.03 p-mentha-1,8-dien-4-ol1706 35.15 1.75 a-terpineol1722 35.72 0.46 dodecanal (not pure)1755 36.44 1.22 bicyclogermacrene1763 36.76 0.09 naphthalene

1772+ 36.89 1.43 citronellol+decanol(major)1804 37.91 0.20 myrtenol1845 39.06 0.09 trans-carveol1857 39.24 0.01 geraniol1864 39.42 0.02 p-cymen-8-ol1882 39.89 0.04 cis-carveol1896 40.44 0.01 cis-p-mentha-1(7)8 dien-2-ol1945 41.78 0.03 neo-isodihydrocarveol1973 42.43 0.28 dodecanol2008 43.38 0.54 caryophyllene oxide (not pure)2030 43.75 0.41 nethyleugenol2050 44.32 5.55 E )-n ero lid o l2071 44.82 0.14 tumulene epoxide II

2096+2096 45.46 0.58 3lemol+(E)-methyl cinnamate2113 45.96 0.07 3umin alcohol2144 46.62 3.00 spathulenol2148 47.00 0.27 iictamnol

27

R R I*R e ten tio n tim e

(m in u tes)A rea

p ercen tageC om p ou n d

2247 48.96 0.39 ftww-a-bergamotol2255 49.14 0.17 a-cadinol2316 50.54 0.01 caryophylladienol I2324 50.65 0.10 caryophylladienol II2389 51.89 0.04 caryophyllenol I2392 52.39 0.18 caryophyllenol II (not pure)

T o ta l 9 4 .3 3

RRI*-relative retention indices calculated against n-alkanes

The main compounds o f A. arida are the four monoterpenes; limonene (21.79%),

myrcene (16.35%), P-pinene (12.28%), linalool (11.02%) and the sequiterpene

alcohol (E)-nerolidol (5.55%).

limonene myrcene

,OH

linalool P-pinene

Figure 9: Chemical structures of the major compounds identified in A. arida essential oil.

28

Agatkosma capensis (L.) Dummer

1. Common name

Boegoe.


Resprouting shrub to 90 cm, sweetly spice-scented. Flowers in lax terminal clusters,

white, pink and purple. Fruits: 3-chambered. Ovary: usually 3-lobed.

3. Distribution

Slopes and flats on shale, granite or coastal sands, less then often on acid sand. This

species is distributed form Namaqualand to Port Elizabeth (Goldblatt and Manning,

2000).

Figure 10: Geographical distribution of A. capensis.


Three different samples of A. capensis were analyzed by GC-MS. One sample was

harvested from the Gamka Mountains, the second sample from the Rooiberg region in

the Cape Province and the third species was collected from Mossel Bay.

29

Figure 11: Gas chromatography profile o f A. capensis (Gamka).

Figure 12: Gas chromatograpy profile o f A. capensis (Rooiberg).

Figure 13: Gas chromatography profile o f A. capensis (Mossel Bay).

Table 3: GC-MS results of A. capensis (Gamka) - A, A. capensis (Rooiberg) - B and

A. capensis (Mossel Bay) - C.

R R I* R eten tio n tim i A rea p ercen tage(A )

A rea percen tag i (B )

A r ea p ercen ta g e(C )

C om p ou n d

1018 8.15 - 0.01 - methyl-2-methyl-buteryte

1032 8.57 0.64 0.76 0.95 a-pinene

1035 8.71 0.10 0.20 0.20 a-thujene (not integrated)

1076 10.30 - 0.01 0.01 camphene

1118 12.17 1.81 2.08 2.44 (3-pinene

1132 12.86 2.20 7.43 3.40 sa b in en e

1159 14.14 0.12 0.39 0.86 8-3-carene

1174 15.03 14.13 25 .31 2 0 .8 8 m y rcen e

1188 15.68 - 0.14 0.30 a-terpinene

1195 16.15 - 0.01 - dehydro-1,8-cineole

1203 16.65 9 .4 3 9 .6 2 8 .2 3 L im o n en e

1218 17.04 2.97 2.40 1 3 .36 p -p h ella n d ren e

1246 18.23 1.19 3.08 6 .3 9 (Z )-P -oc im en e

1255 18.74 0.04 0.29 0.53 y-terpinene

1266 19.04 0.67 2.03 5 .8 8 (E )-P -oc im en e

1280 19.89 0.58 0.71 1.07 p-cymene

1290 20.42 - 0.62 0.74 terpinolene

1319 21.48 - - 0.02 (E)-2,6-dimethyl-1,3,7-nonatriene

1337 22.20 0.02 0.01 0.18 geijerene

1382 24.21 0.02 0.09 0.22 cw-alloocimene

1391 24.51 - - 0.02 (Z)-3-hexenol

1429 25.98 0.12 - 0.07 perillen

1450 26.79 0.05 0.10 0.02 rraw-linalool oxide (furanoid)

1469 27.49 - - 0.13 trans-1,2-limonene epoxide

1474 27.53 0.03 0.29 - OYmy-sabinene hydrate

1476 27.74 - - 0.01 (Z)-p-ocimene epoxide

1478 27.81 0.02 - - cz's-linalool oxide (furanoid)

1498 28.46 0.04 0.02 0.04 "E)-P-ocimene epoxide

1553 30.43 2 1 .5 4 31 .71 2 6 .7 4 inafoo l

1571 31.02 0.07 - 0.17 fr<ms-p-menth-2-en-1 -ol

1571 31.07 - 0.19 - methyl-citronellate

1591 31.79 - 0.11 0.12 Tiyrcenone

1600 31.97 - - 0.11 3-elemene

1611 32.30 - 1.43 1.58 erpinen-4-ol

1626 32.76 0.03 0.05 0.02 ; (!-methyl-6-methylene-3,7-octadien-2-)1

1638 33.02 0.07 0.04 0.11 w-p-menth-2-en-1 -ol1664 33.94 - - 0.01 trans-pinocarveol

1678 34.32 0.01.

C z's-p-menth-2,8-dien-l-ol

31

R R I* R e ten tio n tim e A r ea p ercen tage (A )

A r ea p ercen ta g(B )

e A rea percentage (C )

C om p ou n d

1682 34.36 - - 0.01 5-terpineol

1687 34.64 3 6 .8 7 2.57 - m e th y l-ch a v ico l

1690 34.65 - - 0.36 cryptone

1700 34.90 - Trace - p-mentha-l,8-dien-4-ol

1706 35.15 1.47 4.14 1.03 a-terpineol

1726 35.75 0.03 0.14 0.02 germacrene D

1743 36.07 - 0.01 - a-cadinene

1744 36.21 - - 0.02 phellendral

1748 36.33 - 0.01 0.01 piperitone

1755 36.47 - 0.28 - bicyclogermacrene

1755 36.47 0.04 - - bicyclogermacrene+ carvone

1758 36.85 - 0.01 - cw-piperitol

1772 36.99 0.31 1.10 0.58 citronellol

1802 37.90 - - 0.03 cumin aldehyde

1808 38.02 - 0.07 - nerol

1830 38.59 0.02 0.03 0.05 2,6-dimethyl-3(E),5(E),7-octatriene-2-ol

1845 39.04 - 0.09 0.03 trans-carveol

1845 39.06 2.24 - - (E)-anethole

1857 39.24 - 0.03 0.14 geraniol

1864 39.41 0.10 0.11 0.09 p-cymen-8-ol

1868 39.67 0.02 0.01 - (E)-geranyl acetone

1860 39.71 - - 0.01 8-epi-dictamnol

1949 41.75 0.09 0.08 - (Z)(3)-hexenyl-nonanoate2030 43.74 0.40 0.51 0.06 methyleugenol

2050 44.30 - 0.07 0.03 (E)-nerolidol

2053 44.39 0.31 - - anisaldehyde

2069 44.82 - 0.01 - germacren-D-4-ol

2096 45.47 - 0.01 elemol

2096 45.57 - 0.51 - (E)-methyl-cinnamate

2113 45.93 0.03 0.01 0.05 cumin alcohol

2144 46.62 0.22 0.11 0.06 spathulenol

2148 46.84 0.08 - 0.11 iictamnol

2187 47.70 Trace - -cadinol

2200 47.87 0.05 - r<mj-methyl isoeugenol

2209 48.05 0.01 - t-muurolol

2219 48.39 0.01 0.06 0.07 cimyrcene-II a

2255 49.11 0.09 0.03 Cj-cadinol

2269 49.53 - 0.02 cimyrcene-II b2353 51.41 0.04 - chavicol

T ota l 9 8 .32 9 9 .1 6 9 7 .5 7

RRI* - relative retention indices calculated against n-alkanes

32

The major compounds of A. capensis (Gamka) were determined as the four

monoterpenes methyl-chavicol (36.87%), linalool (21.54%), myrcene (14.13%) and

limonene (9.43%). The major compounds of A capensis (Rooiberg) were determined

as the four monoterpenes linalool (31.71%), myrcene (25.31%), limonene (9.62%)

and sabinene (7.43%). The six monoterpenes identified in A. capensis (Mossel Bay)

are linalool (26.74%), myrcene (20.88%), P-phellandrene (13.36%), limonene

(8.23%), (Z)-P-ocimene (6.39%) and (E)-P-ocimene (5.88%).

All three samples had the following similar major compounds: linalool, myrcene and

limonene. The differences further are the major compound o f A. capensis (Gamka),

methyl-chavicol is not a major compound of A. capensis (Rooiberg) and A. capensis

(Mossel Bay). Likewise sabinene is a major compound for A. capensis (Rooiberg) but

not for A. capensis (Gamka) and A. capensis (Mosel Bay), and P-phellandrene and(E)-

P-ocimene are major compounds for A. capensis (Mossel Bay) but not for A. capensis

(Gamka) and A. capensis (Rooiberg).

O-Me

P-phellandrene (Z)-P-ocimene (E)-P-ocimene

Figure 14: Chemical structures o f the major compounds identified in A capensis essential oils.

33

Agathosma lanata P.A. Bean

1. Botanical descriptionDense, harsh, rounded shrub to 80cm, branching profusely at ground level, herb-

scented. Flowers in dense, woolly terminal clusters, white. Fruits: 3-chambered.

Ovary: usually 3-lobed.

2. Distribution

Dry rocky upper slopes. Rooiberg and Outeniqua Mountains (Goldblatt and Manning,

2000).

Figure 15: Geographical distribution of A. lanata.


Figure 16: Gas chromatography profile o f A. lanata.

34

4. GC-MS results

Table 4: GC-MS results o f A. lanata.R R I* R eten tio n tim e

(m in u tes)A rea p ercen ta g e C o m p o u n d

1032 8.57 4.01 a-pinene1035 8.70 1.00 a-thujene (not integrated)1076 10.30 0.03 camphene1118 12.20 2 1 .5 7 (5-pinene1132 12.86 10.22 sa b in en e1174 14.98 3 0 .7 0 m yrcen e1203 16.57 4.39 limonene1218 17.00 4.52 P-phellandrene1246 18.32 0.13 (Z)-fS-ocimene1255 18.76 0.01 Y-terpinene1266 19.07 0.44 (E)-p-ocimene1280 19.91 1.31 p-cymene1290 20.42 0.06 terpinolene1337 22.20 0.05 geijerene1429 25.97 0.33 perillen1451 26.88 0.02 |3-thujone1466 27.42 0.03 a-cubebene1474 27.55 0.10 trans-sabinene hydrate1495 28.27 0.01 bicycloelemene1498 28.47 0.04 (E)-P-ocimene epoxide1497 28.69 0.17 a-copane1535 29.64 0.04 P-bourbonene1553 30.40 5 .21 lin a loo l1571 31.01 0.05 trans-p-menth-2-en- l-ol1586 31.36 0.13 pinocarvone1600 31.97 0.11 P-elemene1611 32.30 1.73 terpinen-4-ol1626 32.75 0.06 2-methyl-6-methylene-3J7-octadien-2-ol1638 33.02 0.15 c w-p-menth-2-en-1 -ol1648 33.31 0.19 myrtenal1664 33.94 0.28 trans-pinocarveol1687 34.47 0.51 methyl chavicol1690 34.65 0.58 cryptone1706 35.15 1.41 a-terpineol1726 35.75 0.42 germacrene D1740 36.10 0.61 a-muurolene1755 36.50 0.30 bicyclogermacrene (not integrated)1763 36.76 0.08 naphthalene1773 37.06 1.51 S-cadinene1776 37.30 0.60 gamma-cadinene (not integrated)1804 37.91 0.20 myrtenol1810 38.10 0.12 3,7-guaiadiene1830 38.58 0.03 2,6 dimethyl 3(E),5(E),7 octatriene-2-ol1845 39.06 0.01 trans-carveol1853 39.24 0.21 czs-calamenene1864 39.42 0.09 p-cymen-8-ol1860 39.71 0.02 8-epi-dictamnol1893 40.42 0.05 dodecyl acetate1900 40.62 0.20 spi-cubebol1941 41.52 0.01 a-calacorene-11957 41.90 0.10 cubebol (not integrated)1973 42.43 0.10 lodecanol2030 43.75 0.12 nethyleugenol2069 44.84 0.50 germacrene D-4-ol2080 45.10 0.09 tubenol

35

RRI* Retention time (minutes)

Area percentage Compound

2088 45.27 0.04 1-epi-cubenol2096 45.47 0.35 elemol2113 45.93 0.06 cumin alcohol2144 46.62 2.42 spathulenol2187 47.70 0.37 t-cabinol2209 48.07 0.52 t-muurelol2219 48.31 0.07 8-cadinol2219 48.40 0.10 dimyrcene II a (not integrated)2247 48.96 0.09 tra«j-a-bergamotol2255 49.14 0.17 a-cadinol2269 49.52 0.04 dimyrcene II b

Total 99.18RRI*-relative retention indices calculated against n-alkanes

The major compounds of A. lanata are the two linear monoterpenes myrcene

(30.70%) and linalool (5.21%) and the two biciclic monoterpene hydro carbons (3-

pinene (21.57%) and sabinene (10.22%).

sabinene myrcene linalool p-pinene

Figure 17: Chemical structures o f the major compounds identified in A. lanata

essential oil.

36

Agathosma mundtii Cham, and Schltdl

1. Common nameJakkalspisbos.

2. Botanical descriptionSingle-stemmed; sometimes resprouting, finely velvetly, wiry shrub to lm , foetid.

Flowers in terminal or axillary clusters, white. Fruits: 2-chambered, flatsided. Ovary:

usually 1 - or 2-lobed.

3. DistributionMiddle to upper dry rocky slopes. Distributed from the Witteberg to Humansdorp

(Goldblatt and Manning, 2000).

# Agathosma mundti Cham & Schltdl

Figure 18: Geographical distribution of A. mundtii.


37

Table 5: GC-MS results of A. mundtii.R R I* R e ten tio n tim e

(m in u tes)A rea p ercen ta g e C om pound

1032 8.57 0.26 a-pinene1035 8.71 0.28 a-thujene1118 12.20 4 .6 2 (3-pinene1132 12.86 4 .7 9 sa b in en e1174 14.98 1 0 .2 8 m y rcen e1188 15.65 0.35 a-terpinene1203 16.57 1.55 limonene1218 17.00 0.99 (3-phellandrene1246 18.32 5 .8 4 (Z )-P -o ctm en e1255 18.76 1.01 Y-terpinene1266 19.07 2.11 (E)-(3-ocimene1280 19.91 2.22 p-cymene1290 20.42 0.27 terpinolene1319 21.50 0.04 (E)-2,6 dimethyl 1,3,7 nonatriene1327 21.96 0.02 3 methyl-2-butenol1337 22.20 0.15 geijerene1382 24.22 0.24 c/4-alloocimene1413 25.34 0.05 rosefuran1429 25.97 0.01 perillen1450 26.80 0.05 traiw-linalool oxide (fiiranoid)1474 27.55 0.61 tra/M-sabinene hydrate1478 27.85 0.04 cty-linalool oxide (furanoid)1487 28.20 0.01 citronellal

2 9 .8 2 2 0 .0 0 B P 6 9 M + 172 (u n id en tified )1553 3 0 .4 0 1 9 .19 lin a loo l1571 31.01 0.41 trans-p-menth-2-en- l-ol1604 32.01 0.03 thymolmethylether1611 32.33 9 .6 2 te rp in en -4 -o l (n o t p u re)1638 33.02 0.20 cii-p-menth-2-en-1 -oi1687 34.49 1.09 methyl-chavicol1700 34.86 0.02 p-mentha-1,8-dien-4-ol1706 35.15 1.97.. a-terpineol1722 35.64 0.05 2-undecanol1726 35.75 0.48 germacrene D1755 36.44 0.06 bicyclogermacrene1758 36.50 0.10 cw-piperitol (not integrated)1763 36.76 0.08 naphtalene1772 36.99 0.62 citronellol1797 37.70 0.06 b enzy 1 - i sobutyrate1845 39.05 0.01 (E)-anethoIe1854 39.24 0.15 germacrene B1864 39.42 0.01 p-cymen-8-ol1880 40.09 0.31 benzyl -2-methylbutyrate1902 40.72 0.21 benzyl-isovalerate1916 40.96 0.04 a-agarofuran1949 41.75 0.02 'Z)( 3 )-hexenyi-nonanoate1988 42.86 0.02 2-phenylethyl-2-methylbutyrate2008 43.38 0.13 caryophyllene oxide2030 43.75 0.41 methyleugenol2050 44.32 0.06 Ti)-neroiidol2057 44.42 0.10 edol2096 45.46 0.28 elemol2103 45.72 0.28 guaiol2127 46.28 4.47 10-epi-y-eudesmol2144 46.62 0.20 spathulenol2157 47.04 0.06 5-epi-7-epi-a-eudesmol2184 47.48 0.03 ;w-p-menth-3-en-1,2-diol2209 48.09 0.03 -muurolol

38

R R I* R eten tio n tim e (m in u tes)

A r ea p e r cen ta g e C om p ou n d

2 2 3 2 4 8 .6 2 0 .0 6 bulnesol2 2 4 7 4 8 .9 6 0 .1 0 Irans-a-bergamo to 1 o 12 2 5 7 4 9 .13 0 .1 6 (3-eudesmol2 2 8 2 5 0 .6 4 0 .0 4 (Z)-isoeugenol

T o ta l 96.95RRP-retention indices calculated against n-alkanes

Various GC-MS libraries could not identify the major compound o f A. mundtii. This

compound had a base peak of 69 and a molecular mass o f 172. 76.95% (61

compounds) o f the essential oil composition was identified with the following six

monoterpenes as major compounds: linalool (19 %), myrcene (10 %), terpinen-4-ol

(9.62%), (Z)-P-ocimene (5.84%), sabinene (4.79%) and |3-pinene (4.62%).

terpinen-4-ol myrcene linalool

(B-pinene (Z)-p-ocimene sabinene

Figure 20: Chemical structures of the major compounds identified in A. mundtii

essential oil.

39

Agathosma ovalifolia Pillans


Single-stemmed, rounded shrub to 1.5m, acrid or spice-scented. Flowers in lax

terminal clusters white, red-dotted. Fruits: 2-chambered. Ovary: usually 1- or 2-

lobed.

2. Distribution

Rocky quartzitic upper slopes. This species is distributed from the Swartberg

Mountains to Willowmore (Goldblatt and Manning, 2000).

Figure 21: Geographical distribution of A. ovalifolia.


, ' V , . ............. .. , ' P * ,n , r ^ i —,10.00 15-00 20.00 25.00 30.00 35.00 -40-00 -45.00 50.00 55.00 60. OC

Figure 22: Gas chromatography profile of A. ovalifolia.

40

Table 6: GC-MS results of A. ovalifolia.R R I* R e ten tio n tim e A rea p ercen ta g e C om p ou n d1032 8.58 1.55 a-pinene1035 8.70 0.20 a-thujene (not integrated)1118 12.18 1.18 P-pinene1132 12.87 6 .3 6 sa b in en e1159 14.13 0.03 5-3-carene

1174+1176 14.93 2.57 myrcene+a-phellandrene1183 15.10 0.10 pseudo-limonene1188 15.64 0.39 a-terpinene1203 16.59 4 .3 6 lim on en e1218 17.16 2 0 .8 6 P -p h ellan d ren e1246 18.38 12.23 (Z ) p -oc im en e1255 18.78 0.62 y-terpinene1266 19.11 4 .8 4 (E ) p -ocim en e1280 19.91 0.53 p-cymene1290 20.41 0.17 terpinolene1319 21.49 0.06 (E)-2,6-dimethyl-l,3,7-nonatriene1327 21.98 0.25 3-methyl-2-butenol1337 22.19 0.44 geijerene1382 24.21 0.59 cw-alloocimene1451 26.87 0.03 P-thujone1460 27.07 0.06 2,6-dimethyl-1,3-(E),5(E),7octatetraene1474 27.54 0.07 z+<mr-sabinene hydrate1487 28.04 0.07 isoneroloxide I1495 28.24 0.01 bicycloelemene1498 28.45 0.01 (E)-P-ocimene epoxide1553 30.55 0.67 linalool1571 31.04 0.25 hvms-p-menth-2-en-1 -ol1591 31.48 0.02 pregeijerene1611 32.30 2.69 terpinen-4-ol1638 33.02 0.13 cw-p-menth-2-en-1 -ol1655 33.67 0.01 2,6-dimethyl-5-hepten-1 -ol1661 33.81 0.03 frarw-pinocarvyl acetate1682 34.38 0.05 5-terpineol1687 34.40 0.10 methyl-chavicol (not integrated)1690 34.63 0.12 cryp tone1706 35.15 0.30 a-terpineol1720 35.30 0.20 rra«.y-sabinol1726 35.73 0.19 germacrene D1755 36.44 0.40 bicyclogermacrene

1773+1772 37.01 0.46 8-cadinene+citronellol1797 37.68 0.36 benzyl isobutyrate1815 38.07 0.03 2,6-dimethyl-3(E),5(Z),7-octatriene-2-ol1830 38.55 0.18 2,6-dimethyl-3(E),5(E),7-octatriene-2-ol1845 39.04 0.03 fraw-carveol1880 40.07 0.03 jenzyl 2-methylbutyrate1896 40.43 0.06 jhenyl ethyl isobutyrate2030 43.83 1 6 .84 m eth yleu gen ol2098 45.49 0.04 ipobulol2144 46.62 0.91 spathulenol2148 46.80 Trace iictamnol2186 47.65 0.60 sugenol2248 48.878 4 .4 5 d em icin e

T o ta l 8 6 .6 9


41

The major compounds of A. ovalifolia are the five monoterpenes P-phellandrene

(20.86%), (Z)-P-ocimene (12.23%), sabinene (6.36%), (E)-P-ocimene (4.84%) and

limonene (4.36%). The major compounds also include methyleugenol (16.84%) and

elemicine (4.45%).

(Z)-P-ocimene limonene

sabinene

O -M e O -M e

Figure 23: Chemical structures o f the major compounds identified in A. ovalifolia

essential oil.

42

Agathosma ovata Thunb1. Common nameBasterboegoe.

2. Botanical descriptionLeafy shrub, usually single-stemmed to 3m, herb-scented. Flowers auxiliary, white,

pink or purple. Fruits: 5-chambered. Ovary: usually 4- or 5-lobed.

3. DistributionRocky sandstone and silcrete on open slopes and forest margins. This species is

distributed from the Witteberg to Lesotho (Goldblatt and Manning, 2000).

Figure 24: Geographical distribution of A. ovata.


Two different samples o f A. ovata were analyzed by GC-MS. The one sample was

collected form the Gamka Mountains and the other was collected from the Anysberg

region in the Cape.

43

Figure 25: Gas chromatography profile of A. ovata (Gamka).

Figure 26: Gas chromatography profile o f A. ovata (Anysberg).

Table 7: GC-MS results o f A. ovata (Gamka) - A and A. ovata (Anysberg) - B.

R R I* R eten tio n tim e A rea p ercen ta g e (A )

A r e a p ercen ta g e (B )

C o m p o u n d

1017 8.13 0.01 - 4-methyl-2-pentanone1032 8.58 3.39 2.00 a-pinene1035 8.70 1.82 1.29 a-thujene1076 10.30 0.22 0.11 camphene1118 12.24 9 .4 7 4.12 P -p in en e

1132 12.87 15 .68 3 1 .4 3 sa b in en e

1159 14.17 0.02 0.01 5-3-carene1174 15.03 17.18 2.89 m y r ce n e

1176 15.20 - 0.30 a-phellandrene (not integrated)1188 15.63 - 0.23 a-terpinene1195 16.15 0.03 0.01 dehydro-1,8-cineole1203 16.67 4.58 5 .6 4 Iim on en e

1218 17.05 2.32 12.06 P -p h e lla n d ren e

1246 18.31 1.02 0.72 (Z)-(3-ocimene1255 18.76 0.04 1.05 y-terpinene1266 19.08 0.39 0.15 (E)-p-ocimene

44

R R I* R eten tio n tim e A r ea p ercen ta g e (A )

A r ea p ercen ta g e(B)

C om p ou n d

1280 19.91 3.40 6 .91 p -cym en e

1290 20.42 0.03 0.40 terpinolene1319 21.49 0.02 0.02 (E)-2,6-dimethyl-l,3,7-

nonatriene1382 24.22 0.03 0.01 cis-alloocimene1424 25.63 - 0.01 o-methylanisol1429 26.02 0.33 - perillen

1452+1450 26.72 “ 0.04 a-p-dimethylstyrene + trans- linalool oxide (fiiranoid)

1450 26.80 0.36 - trans-Iinalool oxide (fiiranoid)1466 27.40 - 0.48 a-cubebene1474 27.54 0.16 0.16 trans-sabinene hydrate1476 27.72 0.02 - (Z)-(3-ocimene epoxide1478 27.88 0.50 - ci's-linalool oxide (fiiranoid)1487 28.23 0.05 - citronellal1498 28.45 0.10 - (E)-fS-ocimene epoxide1497 28.71 0.03 - a-copaene1505 28.71 - 0.05 dihydroedulene II1532 29.59 0.36 0.27 camphor1553 30.55 17.81 3.08 iinaloo)

1571 31.00 0.43 1.60 rnms-p-menth-2-en-1 -ol1586 31.37 0.04 - pinocarvone1597 31.70 0.41 0.11 bonyl acetate1611 32.30 5.64 13 .64 terp in en -4 -o l

1626 32.76 0.14 “ 2-methyl-6-methylene-3,7 - octadien-2-ol

1638 33.02 0.35 0.66 cw-p-menth-2-en-1 -ol1664 33.06 0.08 - tra/w-pinocarveol1648 33.34 0.17 0.04 myrtenal1651 33.45 - 0.12 sabina ketone1668 34.07 0.37 - citronellyl acetate1687 34.49 2.57 0.15 methyl chavicol1690 34.65 - 1.04 cryptone1690 34.70 0.50 - cryptone (not integrated)1700 34.84 - 0.10 p-menth-l,8-dien-4-ol1706 35.15 1.40 1.44 a-ierpineol1729 35.80 0.27 0.59 cis-1,2-epoxy-terpin-4-ol1733 35.99 0.13 neryl acetate1743 36.07 0.10 a-cadinene (not integrated)1740 36.09 - 0.16 a-muurolene1758 36.85 0.20 0.42 cw-piperitol1763 36.77 0.10 - naphthalene

1772+1773 36.99 1.45 1.58 citronellol+ 8-cadinene1802 37.88 - 0.11 :umin aldehyde1804 37.91 0.14 ]-nyrtenol1830 38.59 0.06

<2,6-dimethyl-3(E),5(E),7-ictatriene-2-ol

1845 39.04 0.06 0.08 rans-carveol1853 39.24 - 0.08 ’w-calamenene

1857+1853 39.24 0.15 ;eraniol+ew-calamenene

45

R R I* R e ten tio n tim e A r ea p ercen ta g e (A )

A rea p ercen ta g e(B )

C om p ou n d

1864 3 9 .4 4 0.23 0 .3 4 p-cym en -8-o l

1900 40.61 0.01 0 .0 6 ep i-cu bebol

1949 4 1 .75 0.13 - (Z )(3 )-h exen y 1-nonanoate

2 0 0 8 4 3 .3 8 - 0 .1 3 caryophyllene ox id e

2 0 3 0 4 3 .7 4 0 .0 6 - m eth yleu gen ol

2 0 5 0 4 4 .3 0 0 .1 0 - (E )-nero lido l

2 0 8 0 4 5 .1 0 - 0 .05 cubenol

2 0 8 8 4 5 .2 6 0 .0 2 0 .0 2 1-ep i-cu benol

2113 4 5 .95 0 .0 7 0 .1 6 cum in a lcoh ol

2 1 4 4 4 6 .6 2 2 .0 4 1.23 spathulenol

2 1 8 4 4 7 .5 0 0.13 0 .2 4 c is-p -m en th -3 -en -1 ,2 -d io l

2 1 8 7 4 7 .7 0 0 .1 9 0 .1 6 t-cad inol

2 2 0 9 4 8 .0 7 0 .3 2 0 .2 6 t-m uurolol

2 2 1 9 48.31 0 .0 6 0 .0 5 5-cad inol

2 2 4 7 4 8 .9 7 0 .03 0 .03 P w u -a -b erg a m o to l

2 2 5 5 4 9 .1 2 0 .8 2 0 .4 8 a-cad inol

T ota l 9 8 .3 4 9 8 .5 7

RRI* - relative retention indices calculated against n-alkanes

Two samples o f A. ovata were analyzed and some differences were noted in their

composition. Agathosma ovata (Gamka) contains the five monoterpenes linalool

(17.81%), myrcene (17.18%), sabinene (15.68%), (3-pinene (9.47%) and terpinen-4-ol

(5.64%) as main compounds. Agathosma ovata (Anysberg) contains the five

monoterpenes sabinene (31.43%), terpinen-4-ol (13.64%), P-phellandrene (12.06%),

p-cymene (6.91%) and limonene (5.64%) as main compounds.

£ 5 $terpinen-4-ol myrcene linalool P-phellandrene

$P-pinene limonene sabinene p-cymene

Figure 27: Chemical structures o f the major compounds identified in A. ovata

essential oil.

46

Agathosma recurvifolia Sond

1. Common name:Kanferboegoe.

2. Botanical descriptionSingle-stemmed, stiff, spreading shrublet to 1.5m, turpentine-scented. Leaves

recurving, with hyaline margins. Flowers in terminal clusters, white. Fruits: 2-

chambered. Ovary: usually 1- or2-lobed.

3. DistributionDry middle to upper slopes and valley bushveld ecotone. Distributed from Rooiberg

and Swartberg Mountains to Uitenhage (Goldblatt and Manning, 2000).

Figure 28: Geographical distribution of A. recurvifolia.


Figure 29: Gas chromatography profile of A. recurvifolia.

47

Table 8: GC-MS results o f A. recurvifolia.

R R I* R e ten tio n tim e (m in u tes)

A r ea p ercen ta g e C o m p o u n d

1032 8.58 1.03 a-pinene1035 8.70 0.41 a-thujene1048 8.98 0.07 2-methyl-3-buten-2-ol1118 12.18 5 .2 7 (5-pinene1132 12.87 5 .8 9 sa b in en e1174 14.93 6 .9 7 m yrcen e1203 16.59 6 .55 lim on en e1280 19.91 1.98 p-cymene1327 21.98 0.07 3-methyl-2-butenol1348 22.79 0.01 6-methyl-5-hepten-2-one1398 24.88 0.09 2-nonanone1429 26.01 1.59 perillen

1450+ 26.81 2.58 frOTW-linalool oxide (furanoid)+cw-l,2- limonene epoxide (0.3%)

1474 27.54 0.26 trarcs-sabinene hydrate1478 27.84 2.13 cis-linalool oxide (furanoid)1498 28.45 0.02 (E)-fl-ocimene epoxide1521 29.38 0.16 2-nonanol1553 3 0 .5 5 3 5 .1 7 lin a loo l1571 31.04 0.18 trans-p-menth-2-en-1 -ol1586 31.38 0.15 pinocarvone1600 31.99 0.04 3-elemene1611 32.30 2.80 terpinen-4-ol1638 33.02 0.19 cw-p-menth-2-en-l-ol1648 33.32 0.38 myrtenal1658 33.86 6 .5 0 sa b in y l aceta te1678 34.32 0.06 cw-p-menth-2,8-dien-1 -ol1687 34.49 0.66 methyl-chavicol1698 35.01 0.45 myrtenyl acetate1706 35.15 2.45 a-terpineol1729 35.81 0.33 cis-1,2-epoxy-terpin-4-ol1733 35.98 0.14 neryl acetate1750 36.32 0.18 cis linalool oxide (pyranoid)1751 36.51 0.35 carvone1765 36.85 0.89 geranyl acetate1772 36.99 0.30 citronellol (not integrated)1797 37.69 0.28 p-methyl acetophenone1804 37.91 0.30 myrtenol1804 39.04 0.22 trans-carveol1857 39.24 0.08 geraniol1864 39.44 1.00 p-cymen-8-ol1882 39.90 0.06 cis-carveol1949 41.75 1.88 (Z)(3)-hexenyl-nonanoate2001 43.14 0.61 isocaryophyllene oxide2008 43.41 2.65 caryophyllene oxide2030 43.74 0.24 methyleugenol2050 44.30 0.23 "E) nerolidol2071 44.80 0.16 lumulene epoxide II2144 46.62 1.98 spathulenol2219 48.39 0.06 iimyrcene-II a2255 49.11 0.16 :x-cadinol

T ota l 9 6 .21

RRP-relative retention indices calculated against n-alkanes

48

The following five monoterpenes, linalool (35.17%), myrcene (6.97%), limonene

(6.55%), sabinene (5.89%) and p-pinene (5.27%) form part of the major compounds

in the essential oil of A. recurvifolia. Sabinyl acetate (6.50%) was also one of the

major compounds.

linalool

P-pinene limonene sabinene

Figure 30: Chemical structures o f the major compounds identified in A. recurvifolia

essential oil.

49

Agathosma serpyllacea Licht. ex. Roem. and Schult


Single-stemmed rounded shrublet. Leaves narrow, swollen behind tip and slightly

twisted. Flowers in many, lax terminal clusters, white, pink or purple. Fruits: 3-

chambered.

2. Distribution

Coastal or inland sand or limestone flats and slopes. Piketberg to Humansdorp.

Figure 31: Geographical distribution of A. serpyllacea.


Figure 32: Gas chromatography profile o f A. serpyllacea.

50

Table 9: GC-MS results o f A. serpyllacea.

RRI* Retention time (minutes)

Areapercentage

Compound

1032 8.57 1.10 a-pinene1035 8.70 0.20 a-thujene (not integrated)1118 12.20 2.18 3-pinene1132 12.86 8.10 sabinene1159 14.17 0.33 5-3-carene1174 14.98 11.82 myrcene1203 16.57 5.81 limonene1218 17.02 1.71 3-phellandrene1225 17.65 0.02 (Z) 3-hexenal1246 18.32 0.48 (Z)-P-ocimene1266 19.07 0.34 (E)-3-ocimene1280 19.91 1.85 p-cymene1290 20.42 0.01 terpinolene1319 21.48 0.05 (E),2,6 dimethyl-1,3,7-nonatriene1337 22.20 0.02 geijerene1360 23.26 0.02 hexanol1382 24.22 0.01 cw-alloocimene1424 25.68 0.20 o-methylanisol1429 25.97 0.29 perillen1450 26.79 0.17 trans-linalool oxide (furanoid)1474 27.56 0.18 trans-sabinene hydrate1478 27.84 0.21 cz's-linalool oxide (furanoid)1498 28.47 0.09 (E)-3-ocimene epoxide1506 28.92 0.08 decanal1553 30.43 26.33 linalool1571 31.02 0.16 /ra«s-p-menth-2-en-1 -ol1611 32.30 2.50 terpinen-4-ol1626 32.75 0.03 2-methyl-6-methylene-3,7-octadien-2-ol1638 33.02 0.14 cw-p-menth-2-en-1 -ol1648 33.28 0.01 myrtenal1678 34.34 0.02 c «-p-menth-2,8-dien-1 -ol1690 34.65 0.43 cryptone1706 35.15 2.12 a-terpineol1758 36.52 0.20 cw-piperitol (not pure)1772 36.99 0.38 citronellol1797 37.69 0.02 p-methyl acetophenone1804 37.90 0.06 myrtenol1830 38.58 0.03 2,6 dimethyl 3(E),5(E),7 octatriene-2-ol1845 39.04 0.05 trans-carveol1857 39.27 0.09 geraniol1864 39.42 0.20 p-cymen-8-ol1949 41.76 0.21 'Z)-3-hexenyl nonanoate2030 43.75 0.39 methyleugenol2050 44.31 0.28 E) nerolidol2144 46.62 0.69 spathulenol2148 46.85 0.17 dictamnol2248 48.97 28.84 elemicine

total 98.60RRP-relative retention indices calculated against n-alkanes

The main compounds o f A. serapyllacea were identified as the four monoterpenes

linalool (26.33%), myrcene (11.82%), sabinene (8.10%) and limonene (5.81%).

Elemicine (28.84%) was also one of the main compounds.

51

elemicine myrcene linalool limonene sabinene

Figure 33: Chemical structures o f the major compounds identified in A. serapyllacea

essential oil.

52

Agathosma zwartbergensis Pillans

1. Botanical descriptionSingle-stemmed, tangled dwarf shrublet to 20cm, lemon-scented. Flowers 2-4 in

terminal clusters, pink. Fruits: 5-chambered. Ovary: usually 4- or 5-lobed.

2. DistributionUpper sandstone slopes. This species is restricted to the Swartberg and Kammanassie

Mountains (Goldblatt and Manning, 2000).

Figure 34: Geographical distribution of A. zwarbergensis.


Figure 35: Gas chromatography profile of A. zwartbergensis.

53

Table 10: GC-MS results o f A. zwartbergensis.R R I* R eten tio n tim e

(m in u tes)A r ea

p ercen ta g eC o m p o u n d

103 2 + 1 0 3 5 8 .5 8 1.48 a-p in en e+ a-th u jen e (0.2% )1118 12.20 0.51 (3-pinene1132 13.05 1.82 sabinene1159 14.17 0 .0 2 8-3-carene1174 15.03 1.30 m yrcene1203 16 .67 0.41 lim on en e1218 17.05 0.13 3-phellandrene1255 18.76 0.11 y-terpinene1266 19.08 0 .0 9 (E )-p -oc im en e1280 19.94 0 .1 7 p-cym en e1290 2 0 .4 2 0 .0 5 terp inolene1337 2 2 .2 2 0 .4 4 geijerene1365 2 3 .48 0 .6 2 m elonal1450 26.81 0 .0 2 /ra/u '-linalool o x id e (furanoid)1487 2 8 .5 0 6 4 .7 2 c itr o n e lla l1553 3 0 .55 7 .9 5 lin a loo l1571 3 1 .0 0 0 .5 4 m ethyl citronellate1583 31 .35 1.45 iso p u leg o l1611 3 2 .3 0 0 .5 5 terp in en -4-o l1641 3 3 .06 0.21 m ethylbenzoate1655 3 3 .70 0 .2 4 2 ,6 -d im eth y l-5 -h ep ten -1 -ol1668 3 4 .1 0 5 .7 0 citronelly l acetate1687 3 4 .4 9 0 .2 5 m ethyl chavico l1706 3 5 .15 0 .4 8 a-terp in eol1740 36.31 0 .03 geranial1765 3 6 .8 7 0 .7 4 geranyl acetate1772 3 6 .9 9 3 .8 2 citron e llo l (not pure)1860 3 9 .74 0 .0 6 8-epi-d ictam nol1973 4 2 .4 4 0 .0 5 dodecanol205 0 4 4 .3 0 0 .1 6 (E )-nerolidol21 4 8 4 6 .8 5 0 .6 5 dictam nol

T ota l 9 5 .2 4


The main compound of the essential oil o f A. zwarbergensis is citronellal (64.72%).

citronellal citronellyl acetate citronellol linalool

Figure 36: Chemical structures o f the major compounds identified in A. zwartbergensis essential oil.

54

4. Results and Discussion

4.1 Antimicrobial activityThe antimicrobial results are summarized in Tables 11, 12 and 13. Antibacterial and

antifungul screening was done followed by the determination o f the minimum

inhibitory concentration (MIC) for selected oil samples showing positive

antimicrobial activity in the disc diffusion assay.

Table 11 shows the disc diffusion antibacterial screening results o f all the Agathosma

species studied. The results were taken after 24 hours of incubation. Zones of

inhibition were measured in millimeters from the edge of the disc containing the

essential oil. All the species studied showed a variable degree o f antibacterial activity.

Agathosma capensis (Mossel Bay) showed activity against E. coli, E. faecalis and S.

aureus. Figure 37 shows a broadscreening of E. coli on all Agathosma oils.

Agathosma lanata and A. zwartbergensis only showed antibacterial activity against B.

cereus. Figure 38 shows the zones of inhibition on selected oil samples (34 - A.

capensis (Gamka); 3 6 - A. ovata (Gamka); 38 -A . zwartbergensis; 40 -A . ovalifolia)

for B. cereus. The A. capensis and the A. ovata samples that were both collected from

the Gamka Mountains in the Cape and Agathosma serpyllacea showed activity against

all test organisms except P. aeruginosa. The A. capensis (Rooiberg) sample showed

little activity against E. coli, S. typhimurium and S. aureus', A. recurvifolia showed

activity against E. coli, E. faecalis and S. aureus', A. ovalifolia showed activity against

E. faecalis and to a lesser extend S. aureus and A. arida showed activity against S.

typhimurium and B. cereus. Agathosma mundtii showed activity against E. faecalis,

S. typhimurium, S. aureus and some initial activity against B. cereus. The activity of

A. mundtii against B. cereus was difficult to determine as initial zones were detected

after 24 hours but regrowth was noted after 48 hours, thus indicating that the essential

oil probably evaporated.

The A. ovata (Anysberg) sample has minimal broad-spectrum antibacterial activity

and was the only species that showed activity against P. aeruginosa.

The largest zone o f inhibition was 7.0 mm from the disc and was the result of the

essential oil o f A. recurvifolia's activity against E. faecalis. Neomycin 30 pg, (Oxoid)

55

was used as a positive control and measured 3.0 mm from the disc, therefore the

inhibition o f the essential oil o f A. recurvifolia was greater than that o f the control.

The antifungal results were similar to the antibacterial results. A small range o f fungi

were however tested. Table 12 shows the antifungal screening results o f all

Agathosma species studied. All the species showed activity against C. neoformans,

with A. arida exhibiting the largest zone (3.0 mm) o f inhibition. No activity was

noted for both C. albicans and A. niger for A. arida. The following species showed

antifungal activity against C. albicans'. A. ovata (Gamka), A. zwartbergensis, A.

ovalifolia, A. recurvifolia and A. serpyllacea. Agathosma ovalifolia was the only

species showing some minimal activity against A. niger.

The minimum inhibitory concentration (MIC) o f the essential oils on the test bacteria

was determined by using the p-iodonitrotetrazolium violet (INT) microplate method.

The MIC o f A. ovata (Gamka and Anysberg samples), A. recurvifolia and A. capensis

(Gamka sample) were determined on E. coli, S. aureus and E. faecalis. The results as

summarized in Table 13 and in Figure 39. These results reflect that the concentration

o f A. capensis (Gamka) oil needed to inhibit the growth o f E. coli is 16 mg/ml, S.

aureus is 32 mg/ml and E. faecalis is 32 mg/ml. The concentration o f A. ovata

(Gamka) oil needed to inhibit the growth oiE . coli is 16 mg/ml, S. aureus is 8 mg/ml

and E. faecalis is 16 mg/ml. The concentration o f A. ovata (Anysberg) oil needed to

inhibit the growth o f E. coli is 8 mg/ml, S. aureus is 8 mg/ml and E. faecalis 16

mg/ml. The MIC o f A. recurvifolia is 8 mg/ml for E. coli, 8 mg/ml for S. aureus and

16 mg/ml for E. faecalis. Well 10E on the microplate showed no INT colouring. This

can be attributed to the fact that well 10E was not inoculated with culture (E. coli).

The MIC for A. recurvifolia for E. coli however stays 8 mg/ml.

The bacteriostatic and fungistatic activities o f the essential oils were evaluated by

using the undiluted oils o f the Agathosma species in the screening tests. Quantitative

results were determined by calculating the minimum inhibitory concentration (MIC),

using the serial dilution method. Agathosma ovata (Gamka) had zones o f inhibition

o f less than 1.0 mm on E. coli, 5.0 mm on E. faecalis and less than 1.0 mm on S.

aureus. The final values taken after 24 hours MIC, for A. ovata (Gamka) on the same

bacteria were 16 mg/ml, 16 mg/ml and 8 mg/ml. Agathosma ovata (Anysberg) had

56

zones o f inhibition o f 3.0 mm on E. coli, 2.0 mm on E. faecalis and 1.0 mm on S.

aureus. The MIC for A. ovata (Anysberg) on the same bacteria were 8 mg/ml, 16

mg/ml and 8 mg/ml. Agathosma capensis (Gamka) had zones o f inhibition o f 1.0 mm

on E. coli, 3.0 mm on E. faecalis and 2.0 mm on S. aureus. The M IC’s for A.

capensis (Gamka) on the same bacteria were 16 mg/ml, 32 mg/ml and 32 mg/ml

respectively. Agathosma recurvifolia had zones o f inhibition o f 1.0 mm on E. coli,

7.0 mm on E. faecalis and 1.5 mm on S. aureus. The MIC for A. recurvifolia on the

same bacteria was 8 mg/ml, 16 mg/ml and 8 mg/ml respectively. The MIC results

therefore correlate with what was observed in the disc diffusion screening results.

The TLC bioautographic assay (Figure 40) with the hydrodistilled oil o f A.

zwartbergensis showed one zone o f inhibition. A TLC bioautographic assay o f pure

citronellal was done simultaneously with the assay o f the hydrodistilled oil. As

indicated on figure 40, the main compound o f A. zwartbergensis, the yellow

compound (R f = 0.79) on the TLC vanillin-sulphuric plate, correlates with the

citronellal standard. This compound was also identified with GC-MS as being

citronellal and could be the antimicrobial factor o f the essential oil.

57

T ab le 11: A n tib a cter ia l sc r e e n in g resu lts as e x p r e sse d in th e d isc d if fu s io n a ssa y (m m from d isc e d g e ).

S p e c ie s E scherich ia co li E nterococcus

faecalis

P seudom onas

aeruginosa

Salm onella

typhim urium

B acillus cereus Staphylococcus

aureus

N e o m y c in 3 0 p g , (O x o id ) 5 .0 3 .0 2 .0 4 .0 10 .0 1 0 .0

A. arida R * R * R * < 1 . 0 2 .0 R *

A. capensis (G a m k a ) 1 .0 3 .0 R* 1.0 1.0 2 .0

A. capensis (R o o ib e r g ) 1 .0 R * R* 1.0 R * 1.5

A. capensis (M o s e l B a y ) < 1 .0 2 .0 R* R * R * 2 .0

A. lanata R * R * R* R * 1.0 R*

A. m undtii R * 1.0 R* < 1 .0 R * 1.0

A. ovalifo lia R * 2 .0 R* R * R * < 1 .0

A. ovata (G a m k a ) < 1 .0 5 .0 R* < 1 .0 2 .0 < 1 .0

A. ovata ( A n y s b e rg ) 3 .0 2 .0 1.0 < 1 . 0 1.0 1.0

A. recurvifo lia 1.0 7 .0 R * R * R* 1.5

A. serpyllacea < 1 .0 3 .0 R * < 1 .0 2 .0 2 .5

A. zw artbergensis R * R * R* R * 3 .0 R *

*R = Resistant

58

Figure 37: Disc diffusion plate o f E. coli on the essential oils o f Agathosma species studied.

Figure 38: Disc diffusion plate o f B. cereus on essential oils o f (34 - A capensis (Gamka); 3 6 - A. ovata (Gamka); 38 — A. zwartbergensis; 40 -A . ovalifolia).

i ( 9 1 • • - # * • ; #

i * t * ' •' «r*» * or m,*.- *;**,'*,

Figure 39: MIC results after 24 hours.

Figure 40: TLC bioautographic assay.

Table 12: Antifungal disc diffusion screening results (expressed as mm from disc edge).

Species C andida alb icans C ryp tococcus neo form ans A sp erg illu s n iger

Nystatin (100 iu, Oxoid). 7.0 5.0 7.0

A. arida R* 3.0 R*

A. capensis (Gamka) R* < 1.0 R*

A. capensis (Rooiberg) R* < 1.0 R*

A. capensis (Mossel Bay) R* 2.0 R*

A. lanata R* 2.0 R*

A. m undtii R* <1.0 R*

A. ova lifo lia 2.0 1.0 1.0

A. ovata (Gamka) 1.0 2.0 R*

A. ovata (Anysberg) R* 2.0 R*

A. recurv ifo lia 2.0 2.0 R*

A. serpyllacea 2.0 2.0 R*

A. zw artbergensis 3.0 2.0 R*

*R = Resistant

60

Table 13: MIC results after 30 minutes, 2 hours and 24 hours.

Microplate

column

Test organism Species MIC (mg/ml) after 30

min

MIC (mg/ml) after 2 hours MIC (mg/ml) after 24

hours

1 E schericha co li A. capensis (Gamka) 8 8 16

2 S taphylococcus aureus A. capensis (Gamka) 8 16 32

3 E nterococcus fa e c a lis A. capensis (Gamka) No colouring 16 32

4 E schericha coli A. ovata (Gamka) 4 8 • 16

5 Staphylococcus aureus A. ovata (Gamka) 4 4 8

6 E nterococcus fa e c a lis A. ovata (Gamka) 2 8 16

7 E schericha co li A. ovata (Anysberg) 4 4 8

8 Staphylococcus aureus A. ovata (Anysberg) 2 4 8

9 E nterococcus fa e c a lis A. ovata (Anysberg) 2 8 16

10 E schericha co li A. recurvifo lia 4 4 8

11 S taphylococcus aureus A. recurvifo lia 2 4 8

12 E nterococcus fa e c a lis A. recurvifo lia 1 8 16

61

4.2 Analytical chemistry

TLC plates of all 10 essential oils were developed and detection was made possible with

the use o f spray-reagents namely vanillin-sulphuric acid (Figure 41) and anisaldehyde-

sulphuric acid (Figure 42). For the TLC plate where vanillin-sulphuric acid was used, the

following similarities were seen on the TLC plates of the individual essential oils.

Agathosma mundtii and A. ovalifolia have similar compounds (coloured blue after

development of the plate) with R f = 0.90. There is a consistency between all 10 samples

in the middle region of the TLC plate. This indicates similar compounds in the different

essential oils. Agathosma capensis (Gamka) contains a unique compound (coloured

brown after development of the plate) with R f = 0.90. Agathosma zwartbergensis

contains a unique compound (coloured yellow-brown after development) with R f = 0.79.

A. ovalifolia contains a unique compound (colour yellow on TLC plate) with R f = 0.59

and Agathosma species contains a unique compound (colour pink) with R f = 0.52.

Similar results were obtained with the anisaldehyde-sulphuric acid colour reagent. Thin

layer chromatography is however not very useful when working with complex mixtures

such as essential oils. The purpose of this study was merely to also produce a fingerprint

of the species studied (e.g. A. zwartbergensis) and to visually summarize the immense

variation between the selected species.

The GC-MS results are tabulated under the monographs o f each species and the major

compounds are clearly indicated. Three samples o f A. capensis were analyzed by GC-

MS. The one sample was harvested from the Gamka Mountains, the other sample was

harvested from the Rooiberg region in the Cape Province and the last sample was

collected from Mossel Bay. There are very interesting differences between the

composition o f the three samples and these results support the fact that external factors

may influence the chemical compositions of species. All three samples had the following

same major compounds: linalool, myrcene and limonene. Methyl-chavicol is a major

compound o f A. capensis (Gamka) but not for A. capensis (Rooiberg) and A. capensis

(Mossel Bay). Sabinene is a major compound for A. capensis (Rooiberg) but not for A.

capensis (Gamka) and A. capensis (Mossel Bay). /3-phellandrene, (Z)-/3-ocimene and

62

Figure 41: TLC plate o f Agathosma essential oils. The plate has been treated with vanillin-sulphuric acid.

#

i i * I * i i I * * «

Figure 42: TLC plate o f Agathosma essential oils. The plate has been treated with anisaldehyde-sulphuric acid.

(E)-P-ocimene are major compounds for A. capensis (Mossel Bay) but not for A. capensis

(Gamka) and A. capensis (Rooiberg). It is important to note that one of the major

compounds of A. mundtii with the highest percentage area (20.00%) could not be

identified using GC-MS. This compound had a base peak of 69 and a molecular mass of

172. This compound could not be suitably matched on many GC-MS libraries and

judging by the comprehensive database used it could be a possible new compound.

Two samples of A. ovata were analyzed by GC-MS where the one sample was collected

from the Gamka Mountains and the other sample was collected form the Anysberg region

in the Cape. Both samples contained sabinene and terpinen-4-ol as major compounds.

There were however some differences in the compositions of the two samples that may

be attributed to external factors. Myrcene and linalool are major compounds of A. ovata

(Gamka) but not for A. ovata (Anysberg). Limonene, p-cymene and p-phellandrene are

major compounds of A. ovata (Anysberg) but not for A. ovata (Gamka).

Previous analytical chemistry research has been done on commercial Agathosma species.

These species include A. betulina and A. crenulata. The major compounds in the

essential oil of A. betulina are isomenthone and diosphenol. Other compounds identified

in A. betulina include limonene, menthone, pulegone, terpinen-4-ol and p-menthan-3-on-

8-thiol. Agathosma crenulata contains a less desirable compound namely pulegone (van

Wyk and Gericke, 2000; Bisset, 1994). None of the Agathosma species in this study

contained isomenthone, pulegone, diosphenol, menthone or />menthan-3-on-8-thiol. All

the Agathosma species in this study contained limonene that was found in previous

studies of A. betulina and A. crenulata. All the species studied except the A. capensis

(Gamka) sample contained terpinen-4-ol, which is also found in A. betulina and A.

crenulata.

64

4.3 Antimicrobial activity of main compoundsAll the Agathosma species studied showed some degree of antimicrobial activity.

Literature studied on essential oil containing plant species show similarities in

antimicrobial activity with the compounds found in some of the Agathosma species.

A study was done by Carson and Riley (1995) to examine the antimicrobial activity of

eight individual components of Tea Tree oil. Tea Tree oil is commonly used to treat skin

disorders such as cuts, bums, insect bites and athlete’s foot. Tea Tree oil contains 1,8-

cineole, l-terpinen-4-ol, p-cymene, linalool, a-terpinene, y-terpinene, a-terpineol and

terpinolene. The test organisms included Candida albicans, Enterococcus faecalis,

Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The results of

the study indicated that terpinen-4-ol was active against all test organisms. Linalool and

a-terpineol were active against all the tested organisms except Pseudomonas aeruginosa.

In this study the Agathosma species containing linalool and a-terpineol as main

compounds were also active against all the test organisms except Pseudomonas

aeruginosa. Agathosma mundtii and A. ovata (Gamka and Anysberg) contain terpinen-4-

ol as a main compound. Agathosma ovata (Anysberg) showed activity towards all the

test organisms, A. mundtii showed activity towards all test organisms except E. coli and

P. aeruginosa and A. ovata (Gamka) showed activity towards all test organisms except P.

aeruginosa. Terpinen-4-ol, p-cymene, linalool and a-terpineol are present, not necessarily

as major components, in all the Agathosma species studied.

Cimanga et al. (2002) indicates that Eucalyptus, Aframomum, Ocimum, Cympobogon and

Monodora species, from the Democratic Republic o f the Congo, contain the essential oil

compounds 1,8-cineole, a-pinene and P-pinene, p-cymene, myrcene, y-terpinene, a-

terpineol, limonene, P-terpineol, citronellal, cryptone, phellandrene and thymol. The

results published in the article indicated that these essential oils showed inhibition of

selected bacterial growth. They compared the chemical composition of the essential oils

of Eucalyptus camadulensis and Cympobogon citratus with their antibacterial activity

and found that their activity is related to the high levels of 1,8-cineole, geranial and neral.

65

Similar bacteria were tested in this study of the antimicrobial activity of buchu and

include E. coli, P. aeruginosa and S. aureus. The Agathosma species studied contain

similar essential oil compounds as the plant species from the Congo. All the Agathosma

species studied contain a-pinene, /3-pinene, p-cymene, myrcene, a-terpineol and limonene

as major or minor compounds.

The essential oil o f Phlomis lanata contains the major compounds; a-pinene, limonene

and trans-caryophyllene. Couladis et al. (2000) on the antimicrobial activity and

chemical composition of Phlomis lanata, indicates that the oil had moderate activity

against the bacteria tested and strong activity against the test fungi. Pure limonene and a-

pinene were tested on the same cultures and the results in the article suggest that the

activity of the oil could be largely attributed to these two main compounds o f the oil.

Agathosma capensis (Gamka and Rooiberg), A. ovata (Anysberg), A. recurvifolia and A.

ovalifolia contain limonene as a major compound. Agathosma mundtii, A. ovata (Gamka)

and A. recurvifolia contain a-pinene as a major compound. The essential oils o f these

Agathosma species with the same major compounds as the essential oil o f Phlomis lanata

showed similar activity towards similar test organisms (E. coli, S. aureus, P. aeruginosa

and C. albicans). Agathosma arida also contains limonene and a-pinene but did not

show activity towards these test organisms. It is noteworthy that all the buchu species

studied contain limonene and a-pinene either as a minor or a major compound.

Cobos et al. (2001) investigated the chemical composition and antimicrobial activity of

the essential oil of Baccharis notosergila. The major compounds were a-pinene,

limonene, j8-caryophyllene and spathulenol. They came to the conclusion that essential

oils containing monoterpenes like limonene are more active against gram-positive

organisms and fungi than gram-negative organisms. Agathosma capensis (Gamka and

Rooiberg), A. ovata (Anysberg), A. recurvifolia and A. ovalifolia contain limonene, a

monoterpene, as a major compound and showed activity towards the gram-positive

bacteria Enterococcus faecalis, Bacillus cereus and Staphylococcus aureus.

The antibacterial activity of Eucalyptus essential oils is due to the synergy of citronellol

and citronellal (Zakarya et al, 1993). Agathosma zwartbergensis contains citronellal as a

66

main constituent and citronellol as a minor constituent. Agathosma mundtii, A. capensis

(Gamka and Rooiberg), A. ovata (Anysberg), A. recurvifolia and A. arida contain

citronellol as a minor constituent. Agathosma ovata (Gamka) contains citronellol and

citronellal as minor constituents.

The positioning of functional groups (terpinen-4-ol compared to oterpineol), level of

ring saturation (carvone compared to dihydrocarvone), type of functional group present

and the level o f chain saturation in an acyclic terpenoid (geraniol compared to citronellol)

determine the antibacterial activity of monoterpenes. Small changes in molecular

properties affect the permeation through bacterial outer membranes and therefore the

antibacterial activity (Griffin et al., 2001). Griffin et al. (1999) examined the structure-

activity relationships of terpenoids. Low water solubility, mainly essential oil

compounds containing hyrocarbons and acetates, attribute to the relative inactivity of

essential oils. Furthermore the antimicrobial activity of oxygenated terpenoid containing

essential oils is associated with hydrogen bonding. It is important to note that water

solubility and hydrogen bonding does not account for all the trends in the activity of

essential oils. The activity o f geraniol, nerol and linalool is largely determined by the

presence o f the alcohol functional group on the carbon skeleton o f these acyclic

terpenoids. The hydrogen-bonding capacity and hence activity is illustrated when

comparing the activity of citronellol, inactive towards E. coli, and geraniol, active

towards E. coli. Citronellal, the corresponding aldehyde to citronellol, was also inactive

towards E. coli and can be attributed to the lower solubility (less hydrogen bonding) than

geraniol, linalol and nerol (Griffin et al., 1999).

When comparing the main constituents o f the Agathosma species studied with the main

constituents o f other species showing antibacterial activity, the compounds present in the

greatest proportions are not necessarily responsible for the greatest share o f the total

activity. It is important to consider the less abundant constituents and the possibility of

synergy between components.

67

5. Conclusion

The main objectives of this study were to investigate the possible antimicrobial properties

of a selection of species belonging to the genus Agathosma and to record the essential oil

profiles of these species.

The presence of antibacterial and antifungal activity of the various Agathosma species

were proven in this study. The activity varied amongst the studied species. Agathosma

recurvifolia for E. faecalis had an activity greater than the positive control (Neomycin 30

fig (Oxoid) used. After comparing the results with antimicrobial results o f other essential

oil containing plant species, it was noted that it is important to consider the less abundant

compounds of the essential oils. The compounds in the greatest proportion are not

necessarily responsible for the antimicrobial activity and the possibility o f synergy

between compounds should be considered. A TLC bioautographic assay was conducted

to determine the antimicobial factor o f A. zwarbergensis. Results indicated that

citronellal could possibly be responsible for the observed antimicrobial activity.

The twelve oils were subjected to GC-MS and the profiles were recorded. For all twelve

oils more than 90% of the compounds were identified. However the main compound of

A. mundtii could not be identified and is most probably a new terpenoid. Each species

still has a unique qualitative and quantitative composition. The differences amongst and

within species could possibly be attributed to some external factors that include the

botanical source, the condition of the plant material (fresh or dried) and the isolation

technique (steam distillation or hydrodistillation).

The results of this report can support the use o f these medicinal plants, generally referred

to as ‘Buchu’, as traditional remedies for selected infectious diseases.

68

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B o ta n ic a l In st itu te o f S o u th A fr ica . P retoria .

Iw u M ., D u n c a n A . an d O k u n ji C . 1 9 9 9 . N e w a n t im ic r o b ia ls o f p la n t o r ig in . P e r s p e c t iv e s o n N e w C rop s

an d N e w U s e s . J. J a n ick (E d ), A S H S P re ss , A le x a n d r ia , V A .

J a n sse n A ., S c h e f fe r J. an d B a e rh e im S v e n d se A . 1 9 8 6 . A n tim ic r o b ia l a c t iv ity o f e s s e n t ia l o ils : A 1 9 7 6 -

1 9 8 6 litera tu re r e v ie w . A s p e c ts o f th e te s t m e th o d s . P la n ta M e d ic a 53: 3 9 5 -3 9 8 .

K a ise r R ., L a m p a r sk y D . and S c h u d e l P . 1 9 7 5 . A n a ly s is o f b u ch u l e a f o il . Journal o f A g r icu ltu ra l and

F o o d C h e m istr y 2 3 : 9 4 3 -9 5 0 .

K h a llo u k i F ., H m a m o u c h i M ., Y o u n o s C ., S o u lim a n i R ., B e s s ie r e J. an d E s s a s s i E . 2 0 0 0 . A n tib a c te r ia l and

m o llu s c ic id a l a c t iv it ie s o f th e e s se n t ia l o il o f C hrysanthem um viscidehirtum . F ito te r a p ia 7 1 : 5 4 4 -5 4 6 .

L a w r e n c e B . 1 9 7 6 . R e c e n t p r o g r ess in e sse n tia l o ils : b u ch u o i l . P e r fu m e r an d F la v o r is t 1 : 1 7 ,

M a n g e n a T . an d M u y im a N . 1 9 9 9 . C o m p a ra tiv e e v a lu a t io n o f th e a n tim ic r o b ia l a c t iv it ie s o f e s s e n t ia l o i ls

o f A rtim is ia afra, P teronia incana an d R osm arinus o ffic ina lis o n s e le c te d b a c te r ia a n d y e a s t stra in s.

L e tter s in A p p lie d M ic r o b io lo g y 2 8 : 2 9 1 -2 9 6 . C f , 1' ' ?

N a k a tsu T ., L u p o A ., C h in n J. and K a n g R . 2 0 0 0 . B io lo g ic a l a c t iv ity o f e s s e n t ia l o i l s a n d th e ir c o n s t itu e n ts .

S tu d ie s in N a tu ra l P ro d u cts C h e m istr y V o l 2 1 . A tta -u r-R a h m a n (E d ), E ls e v ie r S c ie n c e s B .V .

P o s th u m u s M ., v a n B e e k T ., C o ll in s N . an d G ra v en E. 1 9 9 6 . C h e m ic a l c o m p o s it io n o f t h e e s se n t ia l o i ls o f

A g a th o sm a betulina, A. crenu la ta a n d an A. betu lina x crenu la ta h yb rid (b u c h u ) . Jou rn a l o f E sse n tia l

O il R e se a r c h 8: 2 2 3 - 2 2 8 .

T z a k o u O ., P ita r o k ili D ., C h in o u I. an d H arva la C . 2 0 0 1 . C o m p o s it io n and a n tim ic r o b ia l a c t iv ity o f the

e s se n t ia l o i l o f S a lv ia ringens. P la n ta M e d ic a 6 7 : 8 1 -8 3 .

70

v a n W y k B -E ., v a n O u d tsh o o r n B . and G er ick e N . 1 9 9 7 . M e d ic in a l P la n ts o f S o u th A fr ic a . B r iz a , P retoria ,

v a n W y k B -E . and G e r ic k e N . 2 0 0 0 . P e o p le ’s P la n ts . B r iz a , P reto ria .

W a tt J. a n d B r e y e r -B r a n d w ijk M . 1 9 6 2 . T h e M e d ic in a l and P o is o n o u s P la n ts o f S o u th e rn an d E astern

A fr ic a . S e c o n d e d it io n . L o n d o n : E and S L iv in g s to n e .

Z ak arya D . , F k ih -T e to u a n i S . and H ajji F. 1 9 9 3 . C h e m ic a l c o m p o s it io n -a n t im ic r o b ia l a c t iv ity r e la tio n sh ip

o f E u ca lyp tu s e s s e n t ia l o i ls . P la n te s M e d ic in a le s e t P h y to th e r a p ie 2 6 : 3 3 1 -3 3 9 .

h ttp ://w w w .n a tu r a la r o m a th e r a p y .c o m /a r 0 2 .h tm ( A c c e s s e d 0 9 .0 8 .2 0 0 2 )

h ttp : //w w w .o ilso f i ia tu r e .c o m .a u /T e a _ T r e e _ O il/h is to r y _ o f_ te a _ tr e e _ o iI .h tm ( A c c e s s e d 0 9 .0 8 .2 0 0 2 )

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